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Nancy Pelosi and her long-time deputy Steny Hoyer have been ordered to leave their workspaces in the US Capitol by acting House Speaker Patrick McHenry.
Both were told that the locks on their office doors would be "re-keyed".
The evictions come after Kevin McCarthy was removed from the chamber's plum post on Tuesday and Mr McHenry was appointed in the interim.
Mrs Pelosi, who is not currently in Washington, criticised the decision as "a sharp departure from tradition".
Republican Congressman Garret Graves announced on Wednesday that her office would be handed over to Mr McCarthy. He said it is supposed to belong to the "preceding speaker".
"Now that she and other Democrats have caused there to be an immediately preceding speaker, she has removed herself from that office... that was a decision that Democrats and Speaker Pelosi made in giving that office to McCarthy," he said, according to Axios.
Mrs Pelosi noted that "with all the important decisions that the new Republican leadership must address", their first act was to evict her from her office.
"Sadly, because I am in California to mourn the loss of and pay tribute to my dear friend Dianne Feinstein, I am unable to retrieve my belongings at this time."
Ms Feinstein, a California senator who served for more than three decades, died aged 90 last week. Her funeral will take place this Thursday.
While it is rare for members of Congress who are not in leadership roles to have offices in the Capitol building, Mrs Pelosi and Mr Hoyer had so-called hideaways as former Speaker and Majority Leader.
Mrs Pelosi noted in her statement that, in line with the tradition, she had given her Republican predecessor office space while she was Speaker.
Both Democrats will, however, retain their regular workspaces in the House office buildings.
Politico, which first reported the eviction demands, said items belonging to Mrs Pelosi were being packed up on Tuesday evening with the help of staff to her successor, Hakeem Jeffries.
An email to Mrs Pelosi - sent less than two hours after the vote to remove Mr McCarthy as Speaker - reportedly says that the interim speaker is "going to re-assign" the space to others.
It is unclear who exactly made the decision.
Although Mrs Pelosi blamed the temporary speaker for the decision, CNN and the New York Times reported that Mr McCarthy was behind the move and intended to take over the space previously used by Mrs Pelosi.
Neither Mr McHenry, Mr McCarthy nor the Republican-led House Administration Committee, which assigns office space, have yet commented on the matter.
On Tuesday, Mr McCarthy became the first-ever sitting US Speaker to be toppled by a no-confidence vote after a right-wing revolt from within his own party.
Eight rebel Republicans voted with every Democratic member in a so-called motion to vacate against the 58-year-old.
In a lengthy news conference after the vote, Mr McCarthy said he would not run for Speaker again.
He also alleged that, when he became Speaker, Mrs Pelosi had promised to "always back [him] up" if he faced a motion to vacate.
But Mrs Pelosi was notably absent from Tuesday's vote, leaving early before Ms Feinstein's funeral, and her party said it would not step in to rescue Mr McCarthy.
In the official congressional record, she announced that she would have voted to remove Mr McCarthy if she had been present.
Mr McHenry, 47, is a close ally to Mr McCarthy and was a key power-broker during his nine-month speakership.
Though he has been floated as a potential permanent successor, it remains unclear when one will be appointed and who can lead a fractious and slim House Republican majority.
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