STS-81 was the fifth of nine planned missions to Mir and the second one involving an exchange of U.S. astronauts. Astronaut John Blaha, who had been on Mir since 19 September 1996, was replaced by astronaut Jerry Linenger. Linenger spent more than four months on Mir. He returned to Earth on Space Shuttle Mission STS-84.

Atlantis carried the SPACEHAB double module providing additional middeck locker space for secondary experiments. During the five days of docked operations with Mir, the crews transferred water and supplies from one spacecraft to the other. A spacewalk by Linenger and one of his Russian cosmonaut crewmates occurred after the departure of Atlantis.

The STS-81 mission included several experiments in the fields of advanced technology, Earth sciences, fundamental biology, human life sciences, microgravity, and space sciences. It was hoped that data would supply insight for the planning and development of the International Space Station, Earth-based sciences of human and biological processes, and the advancement of commercial technology.