As a top White House aide who witnessed many of the events that prompted the House of Representatives to impeach Trump in December, Bolton could provide new evidence about Trump's efforts to pressure Ukraine to investigate a political rival.
Other witnesses during the House impeachment investigation testified that Bolton strongly objected to an effort by Trump's personal lawyer, Rudy Giuliani, to pressure Kiev outside of regular diplomatic channels, with one saying he referred to the arrangement as a "drug deal."
Congressional investigators believe Bolton objected to Trump's decision to delay $390 million in military aid to Ukraine and could elaborate on that, a Senate aide told Reuters.
Bolton's lawyer said in November he could shed new light on White House discussions, but he refused to participate in the House impeachment inquiry while the Trump administration and Congress battled in court for access to witnesses and documentary evidence.
He said he was now willing to cooperate after a judge dismissed the case last week.
"If the Senate issues a subpoena for my testimony, I am prepared to testify," Bolton said in a prepared statement. He declined further comment.
Democrats seized on Bolton's announcement, saying it bolstered their argument that he and three current administration officials should testify when the Senate begins its impeachment trial.
"If any Senate Republican opposes issuing subpoenas to the four witnesses and documents we have requested they would make absolutely clear they are participating in a cover-up," Senate Democratic leader Chuck Schumer said.
Trump's fellow Republicans have resisted that idea, instead seeking a quick trial based on the evidence collected in the House that could lead to the president's expected acquittal before the 2020 presidential election campaign heats up.
In remarks on the Senate floor, Republican Senate Majority Leader Mitch McConnell repeated his proposal that the chamber postpone a decision on whether to call witnesses until after the trial begins.
That could allow Republicans, who control the chamber by a margin of 53-47, to wrap up the trial without hearing from Bolton or other witnesses.
But Democrats would need only four Republicans to side with them to get the majority required to call witnesses.
DIVISIONS ON WITNESSES
Republican senators were divided on whether they should hear from witnesses like Bolton.
Conservatives such as Senator Richard Shelby expressed no interest in hearing from Bolton, while the more moderate Senator Mitt Romney said he would like to hear from the former high-ranking White House official, when asked by CNN.
"Let the House come over and prove their case and let's vote," Shelby said. "I think it will be pretty fast," he said of the upcoming Senate trial.
Senator Rick Scott, from the politically divided state of Florida, said he wanted to defer a decision until House Democrats and the White House present their case.
Senator Cory Gardner, who could face a tough re-election in Colorado in November, sidestepped questions about whether he would vote for a subpoena for Bolton, saying there was no trial yet because the House had not yet sent over the articles of impeachment.
House Speaker Nancy Pelosi, the top Democrat in Congress, has yet to allow the charges against Trump to be submitted to the Senate, putting a hold on any schedule for a trial. The earliest the House could take any action would be on Tuesday when it reconvenes.
The Democratic-led House has charged Trump with abusing his power for personal gain by asking Ukraine to announce a corruption investigation of former Vice President Joe Biden, a leading contender for the Democratic nomination to face Trump in November's presidential election.
It also charged the president with obstructing Congress by directing administration officials and agencies not to cooperate with the impeachment inquiry.
Trump says he did nothing wrong and has dismissed his impeachment as a partisan bid to undo his 2016 election win.
"I think they've lost their minds," Trump said on Rush Limbaugh's radio program, referring to Democrats.
The White House could try to block Bolton from testifying. That dispute would probably be resolved by U.S. Chief Justice John Roberts, who will preside over the Senate trial, or senators themselves, rather than in court, said Ross Garber, a Washington lawyer who has represented Republican governors in impeachment proceedings.