Since the mysterious deaths of a husband and wife in the Medici family, a powerful Italian dynasty that ruled Florence and Tuscany almost uninterruptedly from 1434 to 1737, rumors have swirled about what led to the couple’s untimely demise. Now, scientists believe they have an answer — it wasn’t murder, but malaria.

In 1587, Grand Duke Francesco I de’ Medici and his wife, Bianca Cappello, died within hours of each other after days of agony.

At the time, logic dictated the culprit to be malaria because the couple had shown symptoms of the illness, including a telltale intermittent fever. But rumors of an assassination immediately spread, pointing to Francesco’s younger brother and rival, Ferdinando, as the perpetrator.

Ferdinando was next in line to the throne, but he was at risk of being passed over in favor of Francesco’s illegitimate son, Antonio. What’s more, Ferdinando had visited the grand duke and his wife at their residence just before they fell ill, further bolstering the suspicion that he poisoned them with arsenic to ensure his own rise to power.

The couple fell ill in a Medici villa in Poggio a Caiano, near Florence, an area dotted with marshes and rice fields — ideal habitats for mosquitoes that can carry malaria. Nonetheless, the murder rumors endured, likely aided by the Medici family’s history of murder and assassination attempts.

Since 2004, when exhumation and analysis of skeletal remains began for 49 Medici family tombs as part of the Medici Project, various studies have confirmed malaria as the cause of Francesco’s demise. However, other studies published as recently as 2006 used toxicological investigations to determine that the couple were indeed victims of arsenic poisoning,

A new study led by Serena Tucci, an assistant professor of anthropology at Yale University’s Faculty of Arts and Sciences, and jointly conducted by Yale and the University of Pisa, Tuscany, used DNA extracted from the skeletal remains of Francesco and another one of his brothers, Giovanni, in an attempt to settle the debate once and for all.

“In recent years, we tried to solve this mystery by performing some specific analysis, in particular paleo-immunological analysis, which attested to the presence of malaria in the remains. But the rumors would not stop, because paleo-immunology is not resolutive, and only ancient DNA could give an answer with a high degree of certainty,” said Valentina Giuffra, a professor of history of medicine at the University of Pisa and a coauthor of the study, published in June in the journal iScience.

Paleo-immunology uses antigens, substances that trigger an immune response, or proteins to check for traces of disease in ancient remains. DNA analysis, which is a more recent approach, is more definitive because it looks for direct genetic signatures of a disease.

Giuffra and her colleagues found genetic traces of plasmodium, the parasitic protozoa responsible for malaria, in samples of bone material from Francesco’s ribs. “DNA is certain,” Giuffra said. “It solves the problem and the doubts. I think this is a definitive answer.”

Malaria is one of the great historical killers for humanity, causing 610,000 deaths in 2024 alone, according to the World Health Organization. It manifests with fever, headaches and chills, and its name comes from the medieval Italian phrase “mal aria,” meaning bad air — a moniker derived from the idea that the disease was contracted by breathing foul-smelling air near swamps or stagnant water.

Historical sources supported the assumption that malaria killed Francesco and Bianca, Giuffra said. Documents written by court physicians of the Medici family described symptoms consistent with the disease. They also detailed some treatments administered to the patients, such as bloodletting — deliberate blood withdrawal, which at the time was thought to release a patient from an illness but in fact worsened their condition.

The genetic analysis was performed on small bone samples kept aside when the Medici tombs were opened in 2004 before the rest of the remains were buried again. Scientists could not perform a similar analysis at the time because the technique wasn’t developed enough, Giuffra said.

The new study found not just one, but two species of the malaria parasite — Plasmodium falciparum and Plasmodium malariae — in Francesco’s remains, suggesting he could have been the victim of a double infection. The researchers also analyzed the remains of Cardinal Giovanni de’ Medici, Francesco’s younger brother, who, along with two other family members, died 25 years earlier after a trip to Tuscany’s coast. Malaria was also found in Giovanni’s sample, in the form of a previously unknown strain of Plasmodium falciparum.

“Francesco and Giovanni, a young member of the family, both traveled 25 years apart to areas of Tuscany which were known for malaria,” Giuffra said.

“The court physicians tried to discourage some members of the Medici family from doing these trips, especially in autumn, which was a season particularly favorable for malaria. But they went anyway, and a few days after the trip, they began to develop the first symptoms, including an intermittent fever, which is associated with malaria infection.”

Detecting different species of malaria also helps trace the evolution of the disease. “Our study contributes to filling a historical gap for a time, Renaissance, and space, Central Italy, from which very limited information about the evolution and spread of malaria exists,” said Alexander Ochoa, an associate research scientist at Yale and first author of the study, in an email.

But is there any guarantee that Francesco was not also poisoned?

“Perhaps not,” Ochoa said, “but the genetic evidence presented in our study decreases the margin for speculation.”

Gisella Caccone, a senior research scientist also at Yale and a study coauthor, agrees. “We can say that they had malaria, we cannot say that they were not poisoned as well,” Caccone said in an email.

“It was already assumed at the time that they had malaria, because of the symptoms they had and the fact that they traveled to the malaria-infested swamps in southern Tuscany — if on top of this someone decided to speed up their departure by poisoning them, we will never know. But how likely is it?”

Donatella Lippi, a professor of history of medicine at the University of Florence and coauthor of the 2006 study that supported the assassination hypothesis, said she still believes that Francesco was poisoned. “Contracting malaria does not mean dying from it, and this research supports what I have always maintained,” Lippi, who was not involved with the study, wrote in an email.

In the case of Francesco’s death, she added, records from the Vatican Library mention skin eruptions, fever and swelling — all symptoms of acute arsenic poisoning.

“I believe Francesco I suffered from malaria, but he was poisoned and died of poison. His tomb was opened 300 years after his death; his hands were contracted as if in the throes of agony, and the body was well preserved — arsenic could explain both.”

Giuffra noted that Lippi’s findings are not based on the skeletal remains exhumed from Francesco’s tomb, but from biological tissue found in a different location where some of Francesco’s organs were supposedly placed after an autopsy, according to historical records used by Lippi. Francesco was known to be an alchemist who experimented with chemical substances, which could explain the skin eruptions, Giuffra added.

The study is interesting, both from a historical and an ancient pathogen perspective, said Anne Stone, a Regents Professor in the School of Human Evolution and Social Change at Arizona State University. Stone, who was not involved with the work, suggests that the brothers died because of malarial infection but that toxicological analyses would have to be done to know whether poison also played a role.

“Recovering pathogen DNA from centuries-old human remains is technically very challenging,” said David Caramelli, a professor of anthropology at the University of Florence who didn’t participate in the study, in an email.

“While the study provides evidence consistent with malaria infection, I do not think it definitively settles the long-standing debate over malaria versus poisoning. The presence of pathogen DNA is not necessarily equivalent to demonstrating the cause of death, and genetic evidence should always be interpreted alongside historical, archaeological, and pathological data.”

Nevertheless, Caramelli concluded, the new research represents an important step forward and demonstrates how paleogenomics can contribute to addressing longstanding historical questions.

EDITOR’S NOTE:  This story has been updated with additional information.

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