The judges’ announcement that an earlier acquittal had been overturned was greeted by a shocked silence in the courtroom here.
The ruling, by the Court of Cassation, means that the case against Ms. Knox, 25, and a former boyfriend, Raffaele Sollecito, 29, will be reheard at a new appeals court in Florence either later this year or in early 2014. The two were initially convicted in 2009 in a trial that divided public opinion internationally, but were acquitted by an appeals court jury
18 months ago. Prosecutors and lawyers for Ms. Kercher’s family then challenged the acquittal, a step that is permitted under Italian law.
Ms. Knox, a student at the University of Washington, stayed up till 2 a.m. in Seattle waiting for the outcome, said her lawyer, Carlo Dalla Vedova. “She was sad,” he said. “She believed the nightmare was over.”
The decision opened a further tangled and dramatic chapter in a long-running case whose youthful protagonists, sometimes-lurid detail and courtroom spectacle have fascinated many people in the United States, Britain and the rest of Europe.
But it also highlighted the divide between the legal systems of Italy and the United States, where defendants cannot be tried twice for the same crime after an acquittal. Ms. Knox’s lawyer said she was unlikely to appear for a new trial, but she could be tried in absentia. The Italian authorities could seek to extradite her only if her conviction was upheld in the new trial and confirmed by the Court of Cassation, whose decisions are final.
The Court of Cassation rules on questions of procedure, not on the merits of a case or the presumption of guilt or innocence.
At the time of the killing, Ms. Kercher and Ms. Knox were living in Perugia, north of Rome. Ms. Kercher, an exchange student at the University of Perugia, was killed in her bedroom on the night of Nov. 1, 2007. Her half-naked body was found under a duvet, her throat slit.
Ms. Knox, then a 20-year-old University of Washington student, and Mr. Sollecito, then 23, were arrested days later and convicted of murder in December 2009 in a lower court in Perugia in a case built largely on DNA evidence. Prosecutors had argued that Ms. Kercher had been the victim of a drug-fueled game of rough sex gone awry, involving Ms. Knox, Mr. Sollecito and a second man, Rudy Guede, an Ivorian living in Perugia, who was tried separately and sentenced to 16 years.
Ms. Knox and Mr. Sollecito were each sentenced to 25 years in prison for the crime, and Ms. Knox received an extra year for slander after she falsely accused another man of committing the murder.
But questions were raised during the appeal about the quality of the forensic evidence, as well as the reliability of some witnesses, and the prosecutors’ theory of the crime.
The convictions were overturned on appeal, and the two were released in October 2011, but last year prosecutors filed an appeal with the Court of Cassation. Ms. Knox’s lawyers appealed the charge of defamation, but the ruling on Tuesday upheld it.
In a statement issued by her media advisers within minutes of the announcement, Ms. Knox said it was “painful” to receive the court’s ruling “when the prosecution’s theory of my involvement in Meredith’s murder has been repeatedly revealed to be completely unfounded and unfair.”
Mr. Dalla Vedova said that the legal reasoning for ordering a new trial was expected from the Court of Cassation within 90 days, and that at that point, lawyers would learn “which points of the case will have to be re-examined” in the new appeals trial. “The trial starts from zero,” he said, “and after we see the decisions we will know whether certain witnesses have to be recalled, or evidence retested.”
If the new appeals court upholds the previous conviction and the Court of Cassation confirms it, Mr. Dalla Vedova said, Ms. Knox will have to serve out her sentence. The lawyer said Italian authorities would have to authorize an extradition request and the United States Justice Department would have to approve it.
There is no final ruling in any case until the Court of Cassation has signed off on it, Mr. Dalla Vedova said.
Giulia Bongiorno, a lawyer representing Mr. Sollecito, said in a telephone interview: “The battle continues. In this trial we always had to climb up the mountain.”
A lawyer for the Kercher family, Francesco Maresca, was jubilant.
“This is marvelous,” he said. “I had faith in the Court of Cassation. I was sure it would annul the acquittal.”
Mr. Maresca said the Kercher family had not traveled to Rome for the latest hearings because Arline Kercher, the victim’s mother, was not well. In a statement, Stephanie Kercher, the victim’s sister, said there were still many unanswered questions. “Understanding the truth about what happened that night is all that we can do for her,” she said. As a family, “we still have a long trip ahead of us, but it is the only one that will allow Meredith to rest in peace.”
HarperCollins said it still planned to publish Ms. Knox’s book, “Waiting to Be Heard,” as planned on April 30 and was moving ahead with scheduled TV interviews.
From the start, the case drew intense media coverage. In the United States, the news media frequently portrayed Ms. Knox as a naïve American wrongly caught up in the morass of a dysfunctional Italian legal system. British newspapers covered the case obsessively at every twist and turn, often from the point of view of the anguished Kercher family.
Mr. Sollecito has been living in Verona, Italy, where he is getting a degree in computer engineering. He did not come to the hearing. “He didn’t want to get caught up in this mob scene, he didn’t want to be here,” his father, Francesco Sollecito, said Monday at the courthouse.
On Tuesday, a reporter from Italian Sky TG24 television managed to reach the younger Mr. Sollecito on the telephone. “Right now, I can’t talk,” he said in a shaky voice. “I am sorry, have a good day.”

Alan Cowell contributed reporting from Paris, and Gaia Pianigiani from Rome.

This article has been revised to reflect the following correction:
Correction: April 1, 2013

Because of an editing error, an article on Wednesday about a new trial ordered by Italy’s highest court in the case of Amanda Knox, an American student accused of murdering her roommate, an exchange student at the University of Perugia, in 2007, misstated the principle of double jeopardy as practiced in the United States. Defendants convicted of a crime can be tried again for the same crime if the conviction is reversed on appeal; it is not the case that defendants can never be tried twice for the same crime after a conviction.