A woman's voice, purportedly that of call girl Patrizia D'Addario, replies: "Which big bed... Putin's?" in an apparent reference to Russian Prime Minister Vladimir Putin.
"Putin's," the male voice says, to which the woman replies: "Oh, how sweet... the one with the curtains."
D'Addario has said Berlusconi, 72, has a four-poster bed that Putin has slept in.
The prime minister's lawyer Niccolo Ghedini scoffed at the recording, calling it "totally worthless material, totally implausible and the stuff of invention."
Anyone using the audio will be sued, he added in a statement.
Berlusconi and Ghedini listened to the recording at the prime minister's villa in Arcore, near his native Milan in northwestern Italy, the ANSA news agency reported.
D'Addario is at the centre of an investigation into an entrepreneur from southern Bari facing corruption charges who allegedly paid call girls to spend the night at the prime minister's Rome residence and a villa in Sardinia.
Berlusconi has rebuffed allegations of paying for sex saying he prefers the "pleasure of conquest".
D'Addario has told the Italian media that she was promised 2,000 euros (2,800 dollars) for each of two overnight visits.
She claims to have recorded conversations with the media tycoon and handed the recordings over to the investigation, which does not directly implicate Berlusconi.
Ghedini questioned how material entrusted under seal to the Bari investigation had "entered into the possession" of journalists in the explosive affair.
A spokesman for Berlusconi's People of Freedom party dismissed the audio segments as "a pathetic attempt to resuscitate a media campaign that has already died".
The prime minister's accusers "should just accept that and go on holiday: they obviously need to", he added.
The L'Espresso group publishes the daily newspaper La Repubblica, which has mounted a relentless campaign urging Berlusconi to explain a string of scandals that have dogged him since late April.
It has led calls for Berlusconi to explain his relationship to Noemi Letizia after his attendance at her 18th birthday party prompted his wife to file for divorce.
Every day, the newspaper publishes a set of 10 questions to the self-made billionaire, a list it updated after the call girl investigation opened.
The flamboyant prime minister has dismissed the allegations as "all lies" and politically motivated.
Further fueling a media frenzy, Italian authorities seized hundreds of photos taken at Berlusconi's Sardinian villa.
Some of the pictures were published in the Spanish paper El Pais and showed Berlusconi in his garden surrounded by women. Other photos showed topless women and a man who was totally nude near a pool.
Berlusconi is also under investigation for allegedly misusing his official plane to fly personal guests, including a flamenco dancer and a well-known singer, to his villa.
The scandals have drawn comment from the Roman Catholic Church, which is heavily influential in Italy.
Cardinal Walter Kasper said that "everybody, but above all a head of government" should behave with "seriousness and sobriety".
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