Launch from Cape Canaveral (KSC); landing on Cape Canaveral (KSC).

STS-65 was the mission "International Microgravity Laboratory" (IML-2). The IML-2 payload consisted of more than 80 experiments in microgravity and life sciences, including five life science experiments developed by American researchers. Of these, Ames Research Center sponsored two experiments using newts and jellyfish. At least two of the four adult newts died on the voyage. Kennedy Space Center (KSC) sponsored the PEMBSIS experiment, designed to study plant embryogenesis in microgravity.

Life Sciences Experiments and facilities on IML-2 include: Aquatic Animal Experiment Unit (AAEU) in Rack 3, Biorack (BR) in Rack 5, Biostack (BSK) in Rack 9, Extended Duration Orbiter Medical Program (EDOMP) and Spinal Changes in Microgravity (SCM) in the Center Isle, Lower Body Negative Pressure Device (LBNPD), Microbial Air Sampler (MAS), Performance Assessment Workstation (PAWS) in the middeck, Slow Rotating Centrifuge Microscope (NIZEMI) in Rack 7, Real Time Radiation Monitoring Device (RRMD) and the Thermoelectric Incubator (TEI) both in Rack 3. Microgravity experiments and facilities on IML-2 include: Applied Research on Separation Methods (RAMSES) in Rack 6, Bubble, Drop and Particle Unit (BDPU) in Rack 8, Critical Point Facility (CPF) in Rack 9, Electromagnetic Containerless Processing Facility (TEMPUS) in Rack 10, Free Flow Electrophoresis Unit (FFEU) in Rack 3, Large Isothermal Furnace (LIF) in Rack 7, Quasi Steady Acceleration Measurement (QSAM) in Rack 3, Space Acceleration Measurement System (SAMS) in the Center Isle, and Vibration Isolation Box Experiment System (VIBES) in Rack 3.

It was the longest Shuttle mission to date.