Information about spawning migration and spawning habitat is essential to maintain and ultimately restore populations of endangered and threatened species of anadromous fish. We used ultrasonic and radiotelemetry to monitor the movements of 35 adult Gulf sturgeon Acipenser oxyrinchus desotoi (a subspecies of the Atlantic sturgeon A. oxyrinchus) as they moved between Choctawhatchee Bay and the Choctawhatchee River system during the spring of 1996 and 1997. Histological analysis of gonadal biopsies was used to determine the sex and reproductive status of individuals. Telemetry results and egg sampling were used to identify Gulf sturgeon spawning sites and to examine the roles that sex and reproductive status play in migratory behavior. Fertilized Gulf sturgeon eggs were collected in six locations in both the upper Choctawhatchee and Pea rivers. Hard bottom substrate, steep banks, and relatively high flows characterized collection sites. Ripe Gulf sturgeon occupied these spawning areas from late March through early May, which included the interval when Gulf sturgeon eggs were collected. For both sexes, ripe fish entered the Choctawhatchee River significantly earlier and at a lower water temperature and migrated further upstream than did nonripe fish. Males entered the Choctawhatchee River at a lower water temperature than females. Results from histology and telemetry support the hypothesis that male Gulf sturgeon may spawn annually, whereas females require more than 1 year between spawning events. Upper river hard bottom areas appear important for the successful spawning of Gulf sturgeon, and care should be taken to protect against habitat loss or degradation of known spawning habitat.