The Court of Cassation rejected a request by defense lawyers for the release of American university student Amanda Knox; her Italian ex-boyfriend, Raffaele Sollecito; and Ivory Coast national Rudy Hermann Guede.
"All three requests have been rejected," said Marco Brusco, a lawyer for Sollecito.
The three are being held in connection with the death of Meredith Kercher, a 21-year-old student from Leeds University in England who was enrolled for a year of study in Perugia, about 110 miles north of Rome.
Kercher was found dead in November in the apartment she shared with Knox. She died from a stab wound to the neck.
Prosecutors have said she was killed resisting sexual assault, and they are investigating the three suspects on suspicion of murder and sexual violence.
Knox, 20, and Sollecito, 24, have been jailed since Nov. 6. Guede, 21, was arrested in Germany on Nov. 20 and later extradited to Italy. He is believed to have fled shortly after the slaying.
All three deny wrongdoing.
The Court of Cassation in Rome did not examine evidence but focused only on whether proper procedures were followed during the investigation, defense lawyers said.
The court's prosecutor called on the judges to reject the defense's appeal, lawyers said during a break in the proceedings.
Judges in Perugia have already rejected defense requests to release the three.
The Perugia judges have ruled that the three could be held for up to a year while the case is investigated. The suspects have not been formally charged.