Columbia launched from Kennedy Space Center on June 5, 1991 at 9:24 AM EDT and landed at Edwards AFB on June 14 at 8:39 AM PDT. The shuttle orbited 146 times at an altitude of 157 nautical miles at an inclination of 39 degrees and travelled 3.8 million miles. The mission lasted 9 days, 2 hours, 14 minutes, and 20 seconds.

This was the fifth Spacelab mission and was dedicated to life science experiments, including humans, 30 rodents and thousands of tiny jellyfish. Primarily the SLS-1 experiments studied six body systems; of 18 investigations, ten involved humans, seven involved rodents, and one used jellyfish.

The six body systems investigated were cardiovascular/cardiopulmonary (heart, lungs and blood vessels); renal/endocrine (kidneys and hormone-secreting organs and glands); blood (blood plasma); immune system (white blood cells); musculoskeletal (muscles and bones); and neurovestibular (brains and nerves, eyes and inner ear). Other payloads included twelve Get Away Special (GAS) canisters installed on the GAS bridge in the cargo bay for experiments in materials science, plant biology and cosmic radiation; Middeck Zero-Gravity Dynamics Experiment (MODE); and seven Orbiter Experiments (OEX).

Columbia was crewed by Commander Brian D. O’Connor, Pilot Sidney M. Guitierez, Mission Specialists James M. Bagian, Tamara E. Jernigan, and M. Rhea Seddon, and Payload Specialists F. Drew Gaffney, and Milly Hughes-Fulford.