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Midfielder Stefano Mauri, 32, was held along with former Genoa midfielder Omar Milanetto, police said.
Juventus coach Antonio Conte, who just led the club to the Serie A title in his first season in charge, is among those being questioned by police.
Officers also visited Italy's pre-Euro 2012 training camp to question left-back Domenico Criscito, 25.
Police are searching more than 30 homes, including those of players, trainers and administrators of clubs in Serie A, Serie B and lower divisions.
For many months now this investigation into match-fixing in the Italian game has been widening and deepening. While up to now much of the suspicion has fallen on teams and players in the lower divisions of the game, the prestigious Serie A is increasingly being drawn into the scandal. None of this will come as any great surprise to those who are following this scandal closely. More painful and damaging, allegations and revelations lie ahead. Of course the fans hate it all, but many will continue to believe that the great bulk of players are clean. And some will argue that at least Italy is trying to tackle the match-fixing issue - while leagues in other countries may be choosing to look away.
Five people were also arrested in Hungary on suspicion of being part of an illegal international betting ring.
Police said Conte was being investigated on suspicion of sporting fraud and fraudulent association over allegations concerning a match between his previous club Siena and Novara in April 2011.
However Conte's lawyer, Antonio De Rencis, said: "Conte's reaction is that of someone who is completely innocent and strongly determined to prove his total innocence."
Later on Monday Italian football federation vice president Demetrio Albertini announced that Cristico, a former Genoa and Juventus defender who is now at Zenit St Petersburg in Russia, would not be selected for Euro 2012 'in order to clear his name'.
"We are disappointed but we have to remain close to the player," Albertini said. "Arrests have been made today but until proven otherwise, we are talking about innocent people even though they are considered suspects.
"I know Domenico and he is incredulous. He is an extraordinary guy and he has already seen his lawyers this morning. It is a strange day for us.
"These are things that we would not like to have had to go through."
Monday's operation was part of a wider investigation which has already seen a number of arrests of current and former Italian players. In June last year, the Interior Ministry set up a special match-fixing task force in response to a number of high-profile cases.
Juventus defender Leonardo Bonucci has also been linked to match-fixing in testimony from a probe in Bari, where he used to play, but is not under formal investigation and will go to the tournament in Ukraine and Poland.
Former Atalanta captain and Italy midfielder Cristiano Doni was banned for three-and-a-half years in August for his part in the 'Calcioscommesse' scandal involving Serie B matches last season. He was also arrested in December 2011 over match-fixing and betting allegations.
Atalanta, promoted from Serie B, were deducted six points in the top flight this season as a result of the scandal.
Former Lazio and Italy striker Giuseppe Signori was banned for five years and 15 other players were banned for between one and five years for their parts.
Mauri, who joined the Rome club from Udinese in 2006 and has 11 caps for Italy, made 16 league appearances last season as Lazio finished fourth in Serie A.
Milanetto, 36, signed for Serie B side Padova last summer after five years at Genoa and made 15 starts last season.
The captain of Lazio has been arrested by police investigating claims of match-fixing.