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Congresswoman Marjorie Taylor Greene of Georgia announced on Wednesday that she will force a vote to remove Mike Johnson as speaker of the House.
The conservative firebrand filed the motion last month but held off on formally introducing it, which would require a vote on Mr Johnson's fate within two legislative days.
At the time, she described her action as a warning to the Republican leader.
Now she has followed through on that threat.
Speaker Johnson put out a statement that her motion "is wrong for the Republican conference, wrong for the institution, and wrong for the country".
Mr Johnson's support for a $61bn military aid package to Ukraine, approved by the House on 20 April, is partially responsible for Ms Greene forcing the vote.
"Mike Johnson is not capable of that job," Ms Greene said on Wednesday. "He has proven it over and over again."
On Tuesday, Democratic leaders in the House of Representatives pledged to support Mr Johnson if his leadership was threatened - a show of gratitude for the speaker's co-operation on Ukraine aid, which was a priority for Democrats and President Joe Biden.
Ms Greene called the Democratic pledge "a warm hug and a big wet sloppy kiss" for the speaker.
Republican leaders, including former president Donald Trump, had reportedly pressured the Georgia congresswoman to back down from attempting to remove Mr Johnson, concerned that a Republican power struggle would harm the party's prospects in November's general election.
Last October, Democrats voted with a handful of Republicans to remove Speaker Kevin McCarthy, setting off three weeks of Republican discord as several top candidates failed to win the near-unanimous party support needed to secure the gavel.
Mr Johnson ultimately prevailed - in part because he was relatively unknown and had few connections to party leadership. But some Republicans, including Ms Greene, soured on him after he worked with Democrats to pass government funding legislation earlier this year.
"I voted for Mike Johnson because his voting record before he became speaker was conservative," Ms Greene said on Wednesday. "Once he became speaker, he has become a man that none of us recognise."
Two other Republicans in the House have said they will back Ms Greene's effort, which would be enough in the narrowly divided chamber to remove the speaker with Democratic help. Instead, the Democrats have said they will vote with pro-Johnson Republicans to "table" the motion, effectively killing.
Ms Greene said she will introduce her motion to remove Mr Johnson next week.