Launch from Cape Canaveral (KSC); landing on Edwards AFB. Columbia was initially scheduled to launch in late February however this date slipped to early March due to concerns with the tip-seal retainers in the main engines' oxidizer turbopumps. All three turbopumps were replaced at the pad but later inspection revealed the retainers to be in good condition. The launch attempt on March 22, 1993 was aborted automatically at T-3 seconds when computers detected an incomplete ignition of the number three SSME. The problem was traced to a leak in the liquid oxygen preburner check valve. All three SSMEs were replaced as a precaution. Another launch attempt on April 24, 1993 was scrubbed due to a possible faulty reading with one of the inertial measurement units.

Columbia carried to orbit the second reusable German Spacelab on the STS-55 mission and demonstrated the shuttle's ability for international cooperation, exploration, and scientific research in space. The Spacelab Module and an exterior experiment support structure contained in Columbia’s payload bay comprised the Spacelab D-2 payload. (The first German Spacelab flight, D-1, flew Shuttle mission STS-61A in October 1985.) The U.S. and Germany gained valuable experience for future space station operations. The D-2 mission, as it was commonly called, augmented the German microgravity research program started by the D-1 mission. The German Aerospace Research Establishment (DLR) had been tasked by the German Space Agency (DARA) to conduct the second mission. DLR, NASA, the European Space Agency (ESA), and agencies in France and Japan contributed to D-2's scientific program. Eleven nations participated in the experiments. Of the 88 experiments conducted on the D-2 mission, four were sponsored by NASA. The crew worked in two shifts around-the-clock to complete investigations into the areas of fluid physics, materials sciences, life sciences, biological sciences, technology, Earth observations, atmospheric physics, and astronomy. Many of the experiments advanced the research of the D-1 mission by conducting similar tests, using upgraded processing hardware, or implementing methods that take full advantage of the technical advancements since 1985. The D-2 mission also contained several new experiments which were not previously flown on the D-1 mission. The D-2 Mission conducted 88 experiments to study life sciences, material sciences, technology applications, Earth observations, astronomy, and atmospheric physics.

One experiment was ROTEX (Robot Technology Experiment on Spacelab D-2-Mission). ROTEX was kind of a starting shot for Germany's participation in space automation and robotics. It contained as much sensor-based on-board autonomy as possible, but on the other side it presumed that for many years cooperation between man and machine, based on powerful telerobotic structures, will be the foundation of high-performance space robot systems, operable especially from ground. The most considered experiment was the autonomous catching of a free-floating object. It was performed to show the capabilities of local feedback loops to remotely control a space robot under communication constraints.

In the amateur radio experiments the crew got contact to the MIR space station.

Due to bad weather at Cape Canaveral the Columbia was diverted to the Edwards AFB.