John Cleese said Thursday the London show is planned as a single event but that further engagements are possible. He also said the group's famous "dead parrot" sketch would likely be incorporated into the performance.
The show will be filmed for possible distribution.
The group had its first big success with the Monty Python's Flying Circus TV show, which ran from 1969 until 1974, winning fans around the world with its bizarre sketches. The group branched out into movies including "Life of Brian" and backed theatrical shows such as "Monty Python's Spamalot."
The veteran comedy troupe - John Cleese, Michael Palin, Eric Idle, Terry Gilliam and Terry Jones - announced the news at a press conference in central London.
Idle said the show would include material that had never been performed live before.
The five surviving members last performed together in 1998. The sixth member, Graham Chapman, died of cancer in 1989.