Nigel Farage has called on former Ukip leader Diane James to stand down as an MEP after she announced she had quit the party to sit as an independent.
Ms James, who was elected an MEP for South-East England in 2014 said her relationship with Ukip had become "increasingly difficult" since she gave up the leadership last month just 18 days after being elected.
Mr Farage denounced the decision as "yet another act of irrational selfishness" and said she was no longer fit to sit as an MEP. Ukip leadership candidate Suzanne Evans also urged her to quit the European Parliament.
In a statement, Ms James said that European Parliament President Martin Schulz had agreed to her request immediately to take up independent status.
Explaining her decision, she said: "At a high profile public event in Cambridge last week, I was asked why I had not completed the process to become leader of Ukip. I had little option but to give the truthful response that, although nominated leader by popular vote in the membership, I found that I had no support within the executive and thus no ability to carry forward the policies on which I had campaigned.
"My decision to retire from the election process and not complete it was very difficult personally and professionally, given that Ukip has dominated my life and all my efforts for over five years.
"In recent weeks, my relationship with the party has been increasingly difficult and I feel it is now time to move on. I wish the party well for the future under new leadership."
Ms James, 56, said she would continue to serve constituents "effectively and diligently" as an independent for the remainder of her five-year term.
But Mr Farage, who returned as interim leader following her decision not to take up the post, said: "This is yet another act of irrational selfishness from Diane James.
"This pattern of behaviour says that is she unfit to continue as an MEP. She should do the honourable thing and resign."
Her departure means that the 24-strong contingent of Ukip MEPs elected in 2014 has been reduced to 20. Amjad Bashir defected to Conservatives in 2015, while Steven Woolfe and Janice Atkinson now sit as independents.
Ms Evans said that all four - including Ms James - should give up their seats, which they secured due to their position on Ukip party lists under a proportional representation system.
"I am very sorry to hear Diane James has decided to leave Ukip," said Ms Evans. "I am sure she has her own personal reasons and I do not question those.
"However she, like Steven Woolfe, were both elected as MEPs on a Ukip ticket, not as individuals. If they have any honour or integrity, both should now stand down as MEPs and vacate their seats. I reiterate my call for defectors Amjad Bashir and Janice Atkinson to do the same, for the same reasons."
Ukip MEP Patrick O'Flynn said: "Sorry to lose a colleague but glad of my judgment that neither she nor Mr Woolfe were suitable candidates for leader."
Ukip is currently undergoing its second leadership contest in a matter of months, with Ms Evans, Paul Nuttall and John Rees-Evans competing for votes of party members, with the successor to Mr Farage due to be announced on November 28.