Gordon Sondland, who donated $1 million to the Republican president's inauguration committee, will meet behind closed doors with staff of three Democratic-led House committees.
The impeachment probe is focusing on a whistleblower's allegations that Trump leveraged nearly $400 million in aid to secure a promise from Ukraine's president to investigate former vice president Biden and his son Hunter, who sat on the board of a Ukrainian energy company.
The House of Representatives Foreign Affairs, Intelligence and Oversight Committees staff are expected to ask Sondland to explain why he became involved in dealings with Ukraine, which is not a member of the European Union.
Sondland was a Seattle-based hotelier until Trump nominated him to his position as ambassador in May. He was confirmed by the US Senate in June and presented his credentials at the European Commission in July.
According to text messages released by House committee leaders last week, Sondland was heavily involved in contacts with Ukrainian President Volodymyr Zelenskiy as he sought a meeting with Trump, and Ukrainian officials expressed concern at the administration's decision to block nearly $400 million in US military assistance for Kiev.
In one of the texts, for example, Sondland emphasized that Trump "really wants the deliverable."
Charges that Trump pressured Zelenskiy in a July 25 telephone call to investigate Biden, a leading rival in Trump's 2020 re-election bid, while withholding the military aid, helped prompt House of Representatives Speaker Nancy Pelosi to announce a formal impeachment investigation last month.
Trump has denied wrongdoing.
Concerns about the call, and possible Trump threats to Ukraine, came to the attention of Congress in a report by a whistleblower. On Sunday, lawyers said a second whistleblower had come forward to substantiate that complaint.
POLITICAL APPOINTEE
Sondland's appearance marks a shift for the investigation because he is a Trump donor and political appointee. Previous witnesses have been career officials, including the former US special envoy for Ukraine, Kurt Volker, and Michael Atkinson, the inspector general of the US Intelligence Community.
Another career diplomat, Marie Yovanovitch, will meet with the committees behind closed doors on Friday. Yovanovitch was the US ambassador to Ukraine until Trump recalled her in May before her term was up, after Trump supporters questioned her loyalty.
The impeachment inquiry has heightened bitter partisan divides in Congress, where Trump's fellow Republicans control the Senate and Democrats have a majority in the House.
Trump has reacted furiously to the inquiry, using obscenities and insulting nicknames for Democratic lawmakers in posts on Twitter. Administration officials - and some Republican allies in Congress - have questioned whether they have any obligation to cooperate with the inquiry.
The White House was expected to tell Pelosi this week that it would ignore lawmakers' demands for documents until the House holds a vote to approve the impeachment inquiry.
Pelosi says a vote is not needed, although Democrats say the House would back the inquiry if there were a vote.
Tuesday's committee meeting with Sondland was to begin at 9:30 a.m. ET (1330 GMT) and could last for hours. Volker was interviewed on Thursday for more than eight hours by House members and staff.
The impeachment investigation could lead to the approval by the House of formal charges against Trump.
A trial on whether to remove him from office would then be held in the Republican-controlled Senate, where Trump continues to enjoy nearly unwavering support from members of his party.