The primary payload of mission STS-35 was ASTRO-1, the fifth flight of the Spacelab system and the second with the Igloo and pallet train configuration. The primary objectives were round-the-clock observations of the celestial sphere in ultraviolet and X-ray spectral wavelengths with the ASTRO-1 observatory, consisting of four telescopes: Hopkins Ultraviolet Telescope (HUT); Wisconsin Ultraviolet Photo-Polarimeter Experiment (WUPPE); Ultraviolet Imaging Telescope (UIT), mounted on the Instrument Pointing System (IPS). The Instrument Pointing System consisted of a three-axis gimbal system mounted on a gimbal support structure connected to a Spacelab pallet at one end and the aft end of the payload at the other, a payload clamping system for support of the mounted experiment during launch and landing, and a control system based on the inertial reference of a three-axis gyro package and operated by a gimbal-mounted microcomputer. The Broad-Band X-Ray Telescope (BBXRT) and its Two-Axis Pointing System (TAPS) rounded out the instrument complement in the aft payload bay.

The crew split into shifts after reaching orbit, with Gardner, Parker, and Parise comprising the Red Team; the Blue team consisted of Hoffman, Durrance, and Lounge. Commander Vance Brand was unassigned to either team and helped coordinate mission activities. The telescopes were powered up and raised from their stowed position by the Red Team 11 hours into the flight. Observations began under the Blue Team 16 hours into the mission after the instruments were checked out. In a typical ASTRO-1 ultraviolet observation, the flight crew member on duty maneuvered the Shuttle to point the cargo bay in the general direction of the astronomical object to be observed. The mission specialist commanded the pointing system to aim the telescopes toward the target. They also locked on to guide stars to help the pointing system remain stable despite orbiter thruster firings. The payload specialist set up each instrument for the upcoming observation, identified the celestial target on the guide television, and provided the necessary pointing corrections for placing the object precisely in the telescope's field of view. He then started the instrument observation sequences and monitored the data being recorded. Because the many observations created a heavy workload, the payload and mission specialists worked together to perform these complicated operations and evaluate the quality of observations. Each observation took between 10 minutes to a little over an hour.