Andrew Mitchell should be sacked by the Prime Minister, shadow home secretary Yvette Cooper said as Labour stepped up its attacks on the embattled chief whip and MP for Sutton Coldfield.
Ms Cooper said the time had passed for clarifications and apologies over Mr Mitchell's alleged use of the word "pleb", and said David Cameron had to act.
Labour has previously raised questions over Mr Mitchell's ability to stay in his post, which he gained in last month's reshuffle, but have stopped short of calling for his resignation or for Mr Cameron to sack him.
Mr Mitchell, who was accused in news reports of a foul-mouthed rant at Downing Street police officers when they would not let him cycle through the street's main gate, has apologised for not treating officers with respect but denied calling them plebs.
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Ms Cooper's call comes ahead of a meeting between Mr Mitchell and the West Mercia Police Federation in his Sutton Coldfield constituency.
She said: "This has gone on long enough. Neither the Prime Minister nor the chief whip have proved capable of coming clean swiftly and putting this right.
"And it is now clear no-one even in the Conservative Party has confidence in Andrew Mitchell either.
"The failure by David Cameron and Andrew Mitchell to take this incident seriously enough and to sort it out straight away means Andrew Mitchell will clearly not be able to instil respect in Parliament or beyond as chief whip, and this will just drag on and on.
"David Cameron needs to put an end to this now and remove Andrew Mitchell from his position as chief whip."
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Mr Mitchell has faced repeated calls for clarification about the incident.
Mr Cameron has insisted that what happened was wrong and should not happen again but has not gone further, highlighting that the officers involved wanted to draw a line under the affair.
Mr Mitchell did not attend this week's Conservative Party conference in Birmingham - despite being the city's only Tory MP - amid speculation that he could yet lose his job.
But Ms Cooper said today: "This problem is just not going to go away because Andrew Mitchell still hasn't come clean about what he did say and the Prime Minister has tried to cover it up.
"We called repeatedly for the Prime Minister to hold a full investigation and for the chief whip to tell the full story about what happened.
"It is completely untenable for the Prime Minister to be unable to mention the police or even to thank them for Olympic security in his annual conference speech because of plebgate.
"And it showed the Prime Minister has not done enough to tackle concern about Government contempt for public servants that the chief whip had to stay away from his own party conference."
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Speaking ahead of today's meeting between Mr Mitchell and officers, chairman of the West Mercia Police Federation Ken Mackaill urged the senior Tory MP to make clear exactly what was said.
On Thursday, he said: "We want to get to the bottom of this matter, hear what Mr Mitchell has to say and hopefully move forward from here.
"Mr Mitchell has apologised for being discourteous to the police officers at Downing Street but has denied using the words attributed to him.
"We want to ask him exactly what he did say. As it currently stands, there is an implication that the officers' accounts are not accurate."
Mr Cameron has called for a line to be drawn under the affair following Mr Mitchell's earlier apologies and senior police figures have also suggested it was time to move on.
But rank-and-file officers remain angry that Mr Mitchell continues to dispute the language the officers reported that he used - most controversially calling them "plebs".
Last month, police officers wearing T-shirts emblazoned with the slogan "PC Pleb and Proud" protested outside Mr Mitchell's constituency office demanding a full inquiry.
Officers from three Police Federation branches, West Midlands, West Mercia and Warwickshire, congregated at the high street base in Sutton Coldfield.
Despite Mr Mitchell's no-show, the "pleb" row continued throughout the conference, with Mr Cameron and other senior politicians repeatedly pressed for further clarity over what was said.
It was also the subject of a stream of jokes throughout the party conference season, with even Association of Chief Police Officers (Acpo) president Sir Hugh Orde apparently unable to resist.
At a fringe meeting at the Tory gathering this week, he is reported to have suggested his organisation be renamed Police Leadership Executive Board - Pleb for short.