Lawmakers who emerged from the deposition declined to provide details from closed-door testimony but they confirmed David Holmes said he had heard the call.
A US official testified Friday that he overheard Donald Trump asking about "investigations" he had requested from Ukraine into domestic political rivals — placing the American president at the center of an alleged conspiracy to extort Kiev.
David Holmes said he heard a phone conversation in a Kiev restaurant in which Trump, on the other end of the line, pressed Washington's EU ambassador Gordon Sondland for information on Ukrainian plans to probe former vice president Joe Biden.
Details of the conversation were included in Holmes's opening statement to impeachment investigators, published by CNN.
The revelation casts doubt on a major plank of the Republican Party's defence against allegations that Trump led a conspiracy to seek foreign help in the 2020 US election.
Republicans have stated that the only direct evidence is the summary of a single July 25 phone call between Trump and his Ukrainian counterpart Volodymyr Zelensky — which they say exonerates the US leader.
Holmes, a career diplomat, said the Trump-Sondland call took place a day later, suggesting that the pressure on Ukraine amounted to a concerted effort rather than just one call.
Sondland told Trump that Zelensky "loves your ass," Holmes is said to have testified.
"I then heard President Trump ask, 'So, he's gonna do the investigation?'" Holmes testified, adding that Sondland replied that Zelensky "will do 'anything you ask him to."
Lawmakers who emerged from the deposition declined to provide details from closed-door testimony, but they confirmed Holmes said he had heard the call.
"It's a matter of public record that Mr Holmes heard this conversation and recognized the president's voice loud and clear because he was so loud on the phone," Democratic congressman Gerry Connolly told CNN.
The call became public knowledge only on Wednesday when William Taylor, the top envoy to Ukraine, testified that Holmes had brought it to his attention.
Holmes, according to his reported opening statement, said he could hear Trump's "very loud and recognizable" voice through the earpiece of the phone.
Even though he did not take notes, Holmes testified that he has "clear recollection that these statements were made."
The New York Times reported that after the call ended, Holmes asked if it was true that Trump did not care about Ukraine.
Sondland replied that Trump cared only about the "big stuff," including investigations pushed by Trump's lawyer Rudy Giuliani because they could benefit him politically.