Finnegan Lee Elder and Gabriel Natale-Hjorth are on trial in Rome over the killing of Mario Cerciello Rega, who was in plain clothes when he was slain in a night drug bust on July 26 last year.
The two face life sentences if found guilty of knowingly killing a police officer.
“This is the latest worrying development to give us the impression they are trying to hide something in this trial,” Elder's lawyer Renato Borzone said.
The prosecutors' office was not immediately available for comment.
Elder, 20, has admitted to stabbing Cerciello with an 8-inch combat knife.
But he insists Cerciello and his partner Andrea Varriale did not identify themselves, and he thought he was fighting for his life against drug dealers.
The San Francisco native, who was 19 at the time of the incident, says Cerciello attacked him from behind, while Varriale wrestled with Natale-Hjorth, then 18.
Varriale says when he and Cerciello stopped the youngsters, they were attacked. Cerciello was left with 11 wounds.
Natale-Hjorth initially told investigators he had not been involved, but his fingerprints were found on a ceiling panel in the hotel room where the students had hidden the knife.
Under Italian law, anyone who participates even indirectly in a murder can face homicide charges.
The defence says lies told by Varriale in the immediate aftermath of the stabbing — such as whether or not the policemen were armed, as they should have been while on duty — seriously undermine his credibility as a witness.
In the latest twist, Elder's lawyers discovered a statement taken during the police investigation was illegally withheld by the prosecution ahead of the trial.
“It's extremely serious, it cannot be considered a mere error,” Borzone said.
In the statement, policeman Fabrizio Pacella admitted drug dealer Italo Pompei was an informant of his.
Pompei was introduced to the Americans by an intermediary, whose bag they stole when they were sold fake drugs.
Borzone said the fact Pompei was a police source could answer many questions surrounding the case, including why Cerciello and Varriale left their designated patrol area, without informing central command, to track down the two young Americans.
Italy's best-selling Corriere della Sera daily suggested the policemen may have been determined to recover the stolen bag because it had a mobile phone inside which could have unmasked Pompei.
“If they've lied about what happened before (the attack), they've lied about what happened during,” Borzone said.
The case continues with three hearings next week, when Pompei and the intermediary will take the stand.
The hearings at the Rome court are being held behind closed doors due to the coronavirus pandemic, despite a nationwide lockdown imposed in March having been nearly entirely lifted.