Democrats who are pursuing an impeachment inquiry against Mr Trump have issued a subpoena to Secretary of State Mike Pompeo for documents concerning contact with the Ukrainian government.
Following a whistleblower complaint that Mr Trump solicited a political favour from Ukraine's president that could help him get re-elected, the lawmakers are pursuing concerns that Mr Trump's actions have jeopardised national security and the integrity of US elections.
The House Foreign Affairs, Intelligence and Oversight Committees, which announced the subpoena, also scheduled depositions for five State Department officials over the next two weeks, including former US Ambassador to Ukraine Marie Yovanovitch, Ambassador Kurt Volker, the US Special Representative for Ukraine, and Ambassador Gordon Sondland, the US ambassador to the European Union.
The State Department did not immediately respond to a request for comment.
In a separate development today, Mr Volker has resigned, US media have reported.
Mr Volker was a key player in US efforts to help resolve an ongoing crisis in Ukraine that started with the annexation of Crimea by Russia and Moscow's support for separatists in the east.
The committees announced the subpoenas after the Trump administration missed a deadline to provide documents and information about contacts with Ukrainian officials, as well as a 25 July telephone call between Mr Trump and Ukrainian President Volodymyr Zelenskiy.
That call is central to an impeachment investigation that Nancy Pelosi, the speaker of the Democratic-led House of Representatives, announced this week.
The impeachment inquiry has cast a new pall over Mr Trump's presidency just months after he emerged from the shadow cast by Special Counsel Robert Mueller's investigation into whether he colluded with Russia in the 2016 election.
Mr Trump has reacted furiously, arguing he did nothing wrong, and accusing Democrats of launching a politically motivated "witch hunt."
The White House this week released a summary of Mr Trump's 25 July phone call where he asked Mr Zelenskiy to investigate his Democratic rival Joe Biden ahead of the November 2020 election - a call at the heart of the whistleblower's complaint.
Mr Trump had ordered $US400 million in military aid and foreign assistance for Ukraine to be put on hold a week before the call, and lifted the freeze earlier this month.
Mr Trump denies he pressured Mr Zelenskiy to do anything improper and said he was not using the aid as leverage, but wanted to make sure Ukraine was taking steps to address corruption issues.
More than 300 former national security officials from both Republican and Democratic administrations on Friday endorsed the House's impeachment inquiry, saying they did not prejudge the outcome but wanted to know more facts.
Ukraine rejects Trump's claims on the Bidens' actions
Meanwhile, a Ukrainian investigation of gas company Burisma is focused solely on activity that took place before Hunter Biden, son of former US vice president Joe Biden, joined its board, Ukraine's anti-corruption investigation agency says.
Separately, a senior official at the general prosecutor's office said that neither of the Bidens had been called for questioning in relation to this investigation.
Ukraine would open an investigation into the period when Hunter Biden was involved with Burisma if there were compelling new testimony in Ukraine, Nazar Kholodnytsky, the head of anti-corruption investigations at Ukraine's Prosecutor's Office, said on Novoye Vremya radio.
But it could not do so on its own initiative, based solely on comments currently being made in the United States, he said.
Ex-foreign minister also dismisses Trump's claims
Ukraine's former foreign minister Pavlo Klimkin has also categorically rejected Mr Trump's claims. Mr Klimkin told the BBC that the prosecutor was sacked for corruption.
A number of Western bodies, including the EU, had pushed for the prosecutor, Viktor Shokin, to be sacked, he said.
Mr Klimkin, who was serving as foreign minister when Mr Shokin was sacked in 2016, said there was "definitely" no evidence that his removal was for anything other than corruption.
"The whole sense of this push was to sort out, to deliver on reforms in Ukraine," he said. "It was not about the prosecutor general. It was about prosecutor offices which were systemically corrupt."
He said it was important to have a full reshuffle of the offices, and pressure was coming from the whole international community, not just Mr Biden or the US individually.
The National Anti-Corruption Bureau of Ukraine (NABU) said an investigation was ongoing into permits granted by officials at the Ministry of Ecology for the use of natural resources to a string of companies managed by Burisma.
But it said the period under investigation was 2010-2012, and noted that this was before the company hired Hunter Biden.
"Changes to the board of Burisma Limited, which are currently the object of international attention, took place only in May 2014, and therefore are not and never were the subject of (the anti-corruption bureau's) investigation," the bureau's statement said.
Hunter Biden was a director on Burisma's board from 2014-2018, according to documents filed by the company in Cyprus, where it is registered.
The investigation into Burisma covers a period when Ukraine was governed by a Kremlin ally, Viktor Yanukovich. Burisma hired Hunter Biden after Mr Yanukovich was toppled in a popular revolt in 2014 and replaced by a pro-Western government.
At the time, many Ukrainian firms were seeking to distance themselves from their relationships with the previous, pro-Moscow authorities, and some invited Western public figures to sit on their boards.
Mr Trump wants Ukraine to investigate the Biden family, accusing Joe Biden of strongarming the Ukrainian authorities, including the then president Petro Poroshenko, to fire general prosecutor Viktor Shokin in 2016 to protect his son.
Mr Shokin says he was fired to prevent him from investigating Hunter Biden, which the Biden family strongly denies.
Mr Kholodnytsky said that were Mr Poroshenko to make a statement about being pressured into removing Mr Shokin from his post, then an investigation must be launched.
However, he said, neither his agency nor the NABU had received such a statement thus far.
Likewise, he said his department couldn't initiate an investigation based solely on comments it had heard from the US side.
"If there was pressure from an official of a foreign country then, first off, the law enforcement agencies of that country must ask what it was."
- Reuters / BBC