“It is pretty obvious to me what is going on,” Mr Johnson told the Parliamentary Press Gallery.
“I think the reality is that Sir Howard started with a short list that did not include much except Heathrow and I think he has been told to have another think.”
Mr Johnson has previously accused the Government of setting up the commission to give it “cover” to reverse its opposition to new runways at Heathrow.
He said on Tuesday that any expansion of Heathrow is not “politically deliverable”.
“I don’t believe it’s going to happen,” Mr Johnson said. “I just don’t see how you can get it through.”
Mr Johnson said that Heathrow is “the wrong way forward for the country” and pledged to fight on to win support for his Thames estuary airport scheme.
“The sooner we get a clear answer from the Government, in my view, the better,” he said. “We can’t keep pussyfooting and fannying around forever.”
He added: “Everybody in my party and indeed in several other parties, so far as I can remember, were elected on a manifesto to oppose a third runway at Heathrow. That happens to be the correct policy. Why change it? Why dump it? The sooner we get back to that, the better.”
Responding to Mr Johnson’s comments, Downing Street said that the Mayor can “speak for himself”, but that the commission should be left to “complete its work”.
Mr Cameron’s official spokesman said: “I will let the Mayor of London speak for himself. It’s important that these big long term decisions are got right. And that’s why it’s right to have this commission. There is a commission looking into this and it should complete its work.”
Sir Howard’s report said that the UK should build a new runway by 2030, with another likely to be needed by 2050.
A final decision on which airport to expand will not be taken until after the 2015 election.
Zac Goldsmith, the Conservative MP for Richmond upon Thames, has suggested he could quit the party if the Government supports expansion of Heathrow.
Mr Goldsmith on Tuesday questioned Sir Howard’s independence and added: “If my party changes its position on Heathrow expansion – the ‘no ifs, no buts, there will be no third runway’ position – if that changes, then yes, I’m obliged to trigger a by-election.”
Sir Howard rejected any suggestions that his commission had been influenced by ministers.
He said: “We have talked to a lot of people and people offered their views, this is a perfectly normal process in the course of producing a report of this kind, but the decisions are ours and made by us alone.”