Republican Matt Gaetz filed a resolution on Monday to remove Mr McCarthy after tensions between the pair flared again when the Speaker passed a bill with the help of Democrats to fund the government and avoid a federal shutdown.
Responding to Matt Gaetz’s move, Mr McCarthy posted on social media: “Bring it on.” Mr Gaetz retorted: “Just did.”
No US Speaker has ever been ousted by a so-called motion to vacate.
The next step for House leaders is to schedule a vote within two legislative days.
If procedural motions do not slow or stop the process, it would take a simple majority of the House to remove Mr McCarthy from his post.
Mr Gaetz has threatened for months to use the procedural tool in a bid to strip his fellow party member of office.
He followed through on those threats by filing a resolution on Monday.
In an earlier speech on the House floor, Mr Gaetz demanded the Speaker disclose the details of a supposed deal he had made with the White House to bring forward legislation to help fund the war in Ukraine during funding negotiations.
He said: “It is becoming increasingly clear who the speaker of the House already works for and it’s not the Republican Conference.”
Brushing off the threat, Mr McCarthy told reporters earlier at the Capitol: “I’m focused on doing the work that has to be done.”
He added that there was “no side deal” on Ukraine, noting he has not spoken to President Joe Biden.
After filing the motion to vacate, Mr Gaetz told a crowd of reporters: “Well, I have enough Republicans where, at this point next week, one of two things will happen.
“Kevin McCarthy won’t be the Speaker of the House, or he’ll be the Speaker of the House working at the pleasure of the Democrats, and I’m at peace with either result, because the American people deserve to know who governs them.”
Asked earlier on Monday whether his actions were plunging the institution into turmoil, Mr Gaetz told reporters: “You talk about chaos as if it’s me forcing a few votes and filing a few motions.
“Real chaos is when the American people have to go through the austerity that is coming if we continue to have $2 trillion annual deficits.”
A motion to vacate is a rare and strong procedural tool which has only been used twice in the past century against Republican speakers.
But in recent years, conservatives have wielded the motion as a potential weapon against their leaders.
Mr McCarthy, hoping to appease some on the hard right like Mr Gaetz as he fought to gain their vote for speaker, agreed in January to give as few as five Republican members the ability to initiate a vote to remove him.
When that was not good enough for his critics, he agreed to reduce that threshold to one - the system that historically has been the norm.
That decision has set Mr McCarthy up for the ultimate test of his leadership as he will now have to rely on Democrats to withhold their support for any effort to force his removal.