The Defenders first appeared as a feature in Marvel Feature #1 (December, 1971), where the founding members gathered to battle the alien techno-wizard Yandroth and then remained as a team afterwards.
However, the origin of the Defenders can actually be traced back to two crossover story arcs originally written by Roy Thomas prior to the "official" founding of the team. The first, in Doctor Strange #183 (November 1969), Sub-Mariner #22 (February 1970), and The Incredible Hulk #126 (April 1970) occurred when the Dr. Strange series was cancelled and the storyline was completed in the other series. Dr. Strange teamed with Sub-Mariner, and then the Hulk to protect the Earth from invasion by Lovecraftian inter-dimensional beings known as the Undying Ones and their leader, the Nameless One. Barbara Norris (later the host body of the Valkyrie) first appeared in this story. In the second arc (featured in Sub-Mariner #34 and #35, Feb. & March 1971), Namor enlists the aid of the Silver Surfer and the Hulk to stop a devastating weather control experiment and to free a small island nation from a dictator, and this led the trio into a face-off with the Avengers.
Although many other heroes worked with the famous "non-team" (so called by the founders because they didn't consider themselves to be an official grouping) in its original incarnation, the most prominent classic Defenders are Doctor Strange, the Hulk, the Sub-Mariner, the Silver Surfer, Nighthawk, and Valkyrie.