Fourteen alleged mobsters have been charged with crimes including 18 murders, in one of Chicago's biggest ever anti-mafia crackdowns.
Joey 'the Clown' and Frank 'the German' have not been found
One of the suspects was found dead on Monday, while two others were said to have escaped arrest.
Two of those indicted were retired policemen, accused of exposing police informers to other mafia members.
"Today the Outfit takes a hit," said US Attorney Patrick Fitzgerald, referring to the branch once run by Al Capone.
All 14 suspects are charged with racketeering, which carries a maximum 20-year prison term.
Eleven are charged with conspiracy to murder, and other counts include extortion, illegal gambling, threats, violence, bribery, intimidation and controlling unions, dating back to 1969.
Two of the murders in question were dramatised in Martin Scorsese's film Casino.
Anthony "the Ant" Spilotro and his brother Michael were killed in Chicago and then buried in a cornfield in the state of Indiana in 1986, Mr Fitzgerald said.
The Spilotro brothers were dumped in a field in Indiana
He pointed out a mistake in the film - Scorsese showed the gangsters being buried alive.
The brothers were apparently suspected of creaming off mafia profits in Las Vegas.
James Marcello, 63, was thought to be the most senior suspect arrested. FBI officials said he was the leader of organised crime in Chicago.
His brother Michael, 55, was also indicted.
They are accused, along with alleged senior mobster Joey "the Clown" Lombardo, 75, and others, of plotting the Spilotro murders and 16 other killings carried out between 1970 and 1986.
Joey "the Clown" and Frank "the German" Schweihs, were still on the run, officials said.
One of the accused, Frank Saladino, 59, was found dead in a hotel room in Chicago, apparently of natural causes.
All the other suspects were either arrested or were already in custody or jail, police said.
The FBI's Robert Grant said the operation made a "significant" impact on organised crime in Chicago.
"This is the first indictment that I can recall that involved so many murders, which really gets at the heart of what [La Cosa Nostra] is... a bunch of murderous thugs," he said.
Mr Lombardo's attorney denied he was involved in the activities, while James Marcello's lawyer did not return calls.