In a posted statement, Bolton said that if called to do so at a Senate trial, "I am prepared to testify."
As a key adviser to Trump, Bolton was close to the president's dealings with Ukraine. The U.S. House of Representatives last month convicted Trump on two articles of impeachment stemming from an effort to pressure the Eastern European nation into launching a politically motivated investigation of Democratic U.S. presidential candidate Joe Biden.
Bolton said during the impeachment hearings he would not testify before the House intelligence committee unless he was first subpoenaed by the committee and then ordered by a judge to defy Trump's wishes by appearing before Congress.
The legal and constitutional struggle over Bolton's possible House testimony went unresolved, he noted in his statement.
"The House has concluded its constitutional responsibility by adopting articles of impeachment related to the Ukraine matter," he said. "It now falls to the Senate to fulfill its constitutional obligation to try impeachments, and it does not appear possible that a final judicial resolution of the still-unanswered constitutional questions can be obtained before the Senate acts.
"Accordingly, since my testimony is once again at issue, I have had to resolve the serious competing issues as best I could, based on careful consideration and study. I have concluded that, if the Senate issues a subpoena for my testimony, I am prepared to testify."
Bolton's credibility as a key member of Trump's foreign policy team and his now-stated willingness to testify at a Senate trial seemed certain to ratchet up pressure on Senate Republican leader Mitch McConnell to strike a deal with Democrats allowing the testimony of witnesses at the trial -- something opposed by most Republicans.
Issuing subpoenas in a trial would require four Republicans to join all the Democrats for a simple majority to pass.
Just when a trial might begin remains uncertain. House Speaker Nancy Pelosi has not yet transmitted the articles of impeachment to the Senate, a move widely seen as a tactic designed to pressure McConnell into allowing witnesses at any trial.
McConnell has called the House's impeachment of Trump "a charade" and said last week that Pelosi's desire to shape the form a Senate trial might take a "fantasy."
Sen. Lindsey Graham, R-S.C., a fervent Trump supporter, suggested Sunday the Senate should change its rules to enable a trial to begin immediately, without the need for the House to transmit the articles of impeachment.
Such a move would need a simple majority of 51 votes. Republicans control 53 of the Senate's 100 seats.