Ms Harman says Mr Brown is the "solution" not the "problem"
Harriet Harman has insisted she is "not preparing the ground for a leadership election" amid growing speculation over Prime Minister Gordon Brown's future.
The Commons Leader rejected a Times report she was overheard telling aides after Labour's Glasgow East by-election defeat: "This is my moment."
Mr Brown has been under pressure since Labour saw its majority overturned by the SNP in last Thursday's poll.
Two Labour MPs have openly called for him to consider resigning.
But Downing Street sources told the Press Association that reports Ms Harman and Foreign Secretary David Miliband were preparing leadership challenges were "garbage".
In a statement, Ms Harman said: "I am not preparing the ground for a leadership election.
"In respect of Labour's defeat in the Glasgow East by-election, I did not tell aides - or anyone else - that 'this is my moment'.
"I was bitterly disappointed by the Glasgow East by-election result in which I campaigned in support of Margaret Curran - a woman who I admire greatly."
She added: "My 'public protestations of loyalty' to Gordon Brown are no different to what I have expressed in private. I do not accept 'it is over'."
There has been continuing speculation about Labour unease with Mr Brown's performance as PM, with Tuesday's London Evening Standard claiming 10 junior ministers may be ready to quit to force Mr Brown to step down.
So far only Labour MPs Gordon Prentice and Graham Stringer have publicly called for a change of leader.
By contrast several leading government figures, including Home Secretary Jacqui Smith, Justice Secretary Jack Straw, Schools Secretary Ed Balls and Cabinet Office Minister Ed Miliband, have publicly defended Mr Brown.
On Sunday, former deputy prime minister John Prescott demanded an end to "pointless" manoeuvring against Mr Brown - and insisted no other minister was "anywhere near" capable of taking over at Number 10.
And Ms Harman, who is "minding the shop" while Mr Brown is on holiday, also insisted on Monday that he was "the solution" and not "the problem".
But Paul Kenny, leader of the GMB union, urged Labour MPs to trigger a ballot ahead of the party's annual conference in September.
Following the by-election result, he said it was "better to have the ballot" and "see where we go".