The Interior Ministry said on Sunday 66.5% of voters had cast their ballots by the close of polling stations across the 110 provinces for the day at 10pm (2000 GMT).
Polls will reopen on Monday at 7am, with the partial results expected within a few hours of the 3pm close.
Analysts say Berlusconi's chances of securing a second term depend on voters largely disenchanted by an economy that has failed to rebound during his five years at the helm.
On Sunday, Corriere della Sera, Italy's leading daily, ran a front-page editorial cartoon depicting Italy as a woman with a clothespin on her nose while she pondered her ballot choice.
In Rome, priests and nuns were among the earliest voters, casting ballots before heading over to St Peter's Square for Palm Sunday mass.
The most recent opinion polls, conducted 10 days before the end of campaigning, gave Prodi - a former prime minister and one-time European Commission president - a slight edge.
Opposition leader Romano Prodi
had an edge in opinion polls
A centre-left candidate, he defeated the conservative Berlusconi in elections in 1996.
Should Prodi win again this time, he is expected to tone down Berlusconi's staunch championing of the US administration.
Berlusconi defied fierce domestic opposition to the invasion of Iraq by sending a contingent of Italian troops following the ouster of Saddam Hussein.
Both candidates have pledged to bring home Italy's troops from Iraq by year's end, although Prodi recently added he would try to do so as soon as possible.
Berlusconi has predicted that a high turnout would propel him to victory.
"Vote! Vote! Vote!" headlined conservative Milan daily Il Giornale, which is owned by Paolo Berlusconi, the premier's brother.
Voting is being spread over two
Italy's richest man, billionaire Berlusconi has a business empire that includes Italy's three main private TV networks, a soccer team, as well as publishing, advertising, insurance and real estate interests.
Critics say he spent most of his premiership pushing through laws to protect his business interests and to help him in his years of judicial woes.
Berlusconi contends the laws benefit all Italians and that he has been the innocent victim of left-leaning prosecutors.
He is hoping that his promises of tax cuts can bring a surprise victory for his Forza Italia party.Heated campaign
However, many Italians say the actual policies of the candidates have been overshadowed by the stream of invective they have flung at each other.
The insults doled out included Prodi likening Berlusconi to a drunkard clinging to a lamppost, while the prime minister retorted that his opponent was a "useful idiot".
A high turnout could make the
result harder to predict
Towards the end of the campaign Berlusconi publicly insulted Italians when, using a vulgar word, he said that only morons would vote for the opposition.
In his final election speech on Friday in Naples, the 69-year-old owner of Mediaset and AC Milan likened his centre-left opponents to Stalin and Pol Pot and said Italy's "Christian identity" was at stake.
His policy pledges have included promises to abolish a homeowner's property tax and raise minimum old-age pensions.
Meanwhile Prodi, who was prime minister from 1996 to 1997 before becoming president of the European Commission, has campaigned on a call for national unity, promising tax breaks for Italian companies and a cut in payroll taxes to try to spur hiring.
The current vote is taking place under a new proportional system, following a law pushed through by Berlusconi's government to increase the chances that his smaller allies would win seats.
That has raised fears of a return to "revolving-door" leadership that Berlusconi ended by becoming the head of Italy's longest serving post-war government in 2001.
There have been 60 administrations since World War II, and coalition instability has often brought down governments within their first year of office.
Voters have to choose one of 74 symbols of parties, many of them overlapping as part of coalitions.
By noon on Sunday, four hours after voting began, 17.6% of eligible voters had cast ballots, the Interior Ministry said. That was down from 21.5% in the 2001 vote, but that year, there was only one day of voting, not two.
Some 47 million Italians are eligible to vote.
And, for the first time in a parliamentary election, Italians living abroad have been eligible to vote.
By the deadline according to officials some 1.1 million Italian expatriates had mailed in their votes.