Sir Sadiq Khan has consulted lawyers over the government's plans for a third runway at Heathrow, which he opposes.
The move puts the mayor of London on a potential collision course with ministers in his own party, after the chancellor backed plans for the expansion of the airport in January.
Rachel Reeves said the airport expansion was vital for kickstarting economic growth and promised to "stop blockers getting in the way of development".
But Sir Sadiq, one of the most senior Labour figures outside of Westminster, has warned about the impact on noise, air pollution and climate change.
Plans to expand Heathrow's runway capacity were first proposed in 2006, but have been the subject of rows ever since.
Earlier this year, Reeves announced she was in favour of the expansion and said she hoped the new runway would be operational by 2035.
The chancellor's support for the project was first reported by Bloomberg News on 20 January.
Information released to BBC News under Freedom of Information laws reveals that Sir Sadiq's officials asked in-house lawyers for legal advice just two days later.
Legal advice was provided to the mayor on 24 January, but it's not known what his lawyers said.
The following week the London mayor said he would "scrutinise carefully any new proposals" but that he was "simply not convinced" the expansion could go ahead without damaging the environment.
A government source acknowledged there was some awkwardness around a senior Labour figure being involved in potential legal battles against government-backed plans, but said the mayor's move was neither surprising nor concerning.
They pointed to the length of time any expansion would take and said "Sadiq probably won't be mayor of London by the time this comes to pass".
Heathrow will submit initial plans for the expansion this summer, with a full proposal likely to follow in the months after.
That proposal would have to be approved by both the Planning Inspectorate and the transport secretary before work gets under way.
Any potential legal action from the mayor of London or others could only officially begin after those approvals.
