The Miami Dolphins are to play the New York Giants in London on 28 October, the NFL has confirmed.
Wembley fought off opposition from Twickenham and Germany
As exclusively revealed by BBC sports editor Mihir Bose earlier this month, the new Wembley will host the match.
It will be the first time a regular season NFL match has been staged outside North America.
NFL owners voted last October to play up to two games outside the USA every year for the next five years, with just one game to be played in 2007.
The NFL staged its first regular-season game outside the United States in 2005 when 103,467 people saw the Arizona Cardinals play the San Francisco 49ers in Mexico City.
That was the largest crowd for a regular-season game in NFL history.
The Dolphins are proud to be a part of such a historic occasion.
Miami owner Wayne Huizenga
NFL Europe failed to take root in Britain, where the London Monarchs and Scottish Claymores eventually folded.
But NFL commissioner Roger Goodell insisted there is still a huge NFL fan base in Britain, with many of the league's supporters having watched the game when American football was broadcast on terrestrial television in the 1980s.
He said: "The international popularity of the NFL grows every year. That interest will be seen as the far corners of the world watch Super Bowl this Sunday.
"That is a fascination we will build upon. London and its international stature adds to the impact of the game."
In the Dolphins and the Giants, the NFL is sending two of its most famous teams across the Atlantic.
Miami owner Wayne Huizenga said: "The Dolphins are proud to be a part of such a historic occasion.
"We were one of the first NFL teams to play in the UK with our American Bowl appearance in London in 1988. Dolphins games always have been among the most popular NFL telecasts in England.
We're looking forward to bringing our team to London, which has long been a centre of attention and enthusiasm for the NFL.
Giants president John Mara
"We have a great following there, including fan clubs throughout the country.
Giants president John Mara added: "We are looking forward to bringing our team to London, which has long been a centre of attention and enthusiasm for the NFL overseas.
"Playing in London in late October means our fans there will see a mid-season contest. It will signal the beginning of the second half of the season when teams begin their play-off run."
Mayor of London Ken Livingstone welcomed the announcement.
"We are expecting nearly 10,000 American fans to travel to London to watch the game as well as people from Europe.
"There will be many millions of TV viewers in the United States alone, so it will be a great opportunity to showcase London to new visitors and for people to see the way we uniquely enjoy our sport."