The Tampa Bay Buccaneers will host the Chicago Bears at Wembley on 23 October, should the 2011 NFL season go ahead.
However, a question mark remains over the game because of the current dispute between team owners and players.
If the fixture is held, it will be the fifth year running that a regular season game has been staged in London.
And the Buccaneers would become the first side to make a second competitive visit to the capital after their defeat by the New England Patriots in 2009.
The Bears previously played in the UK when, as reigning Super Bowl champions, they beat the Dallas Cowboys in a pre-season match at Wembley in 1986.
The NFL owners imposed a "lockout" on 12 March after the collective bargaining agreement with the players expired, and talks over pay and other conditions failed to produce a renegotiated agreement.
There is a danger that the dispute could force the cancellation of the 2011 season, and an NFL statement said that if it is not resolved by 1 August, the Wembley game will instead be played at the Buccaneers' Raymond James Stadium.
"Our past four games in London have demonstrated the tremendous passion for NFL football that exists in the UK," said NFL commissioner Roger Goodell.
"We hope by staging another extraordinary game at Wembley that we can continue to grow our existing fan base and attract even more new fans."
London Mayor Boris Johnson added: "Continuing London's love affair with the hugely popular stateside sport, we are proud to once again welcome two of America's top NFL teams to the capital.
"With a growing local fanbase it is a fantastic opportunity for enthusiasts from London and beyond to experience the unique thrill of a live game in one of the most iconic sports venues around, Wembley Stadium."
The full schedule for the 2011 NFL season is set to be unveiled on Tuesday.