We assess temporal and spatial distribution and diel variability in activity of Gulf of Mexico sturgeon Acipenser oxyrinchus desotoi in the Pensacola Bay system, Florida, using stationary ultrasonic telemetry. Gulf of Mexico sturgeon (n = 54) migrated through the bay system in fall to wintering areas in the Gulf of Mexico and Santa Rosa Sound. In spring, sturgeon migrated back through the bay system to summering habitats in rivers. Gulf of Mexico sturgeon use East Bay and Escambia Bay primarily as migration routes between riverine areas used in spring and summer and the Gulf of Mexico used in winter. North Central Pensacola Bay was not routinely frequented. Gulf of Mexico sturgeon used specific areas within the Pensacola Bay system in summer and winter not previously documented as essential sturgeon habitat. Areas in southeastern Pensacola were used heavily during winter by a portion of the population. Gulf of Mexico sturgeon also exhibited long-term winter residency in Santa Rosa Sound. Interestingly, an area in northeastern Escambia Bay supported Gulf of Mexico sturgeon in summer. This observation was unexpected; however, the identification of Gulf of Mexico sturgeon in this area at this time has important ecological and management implications. Gulf of Mexico sturgeon exhibited a strong diel activity pattern. Gulf of Mexico sturgeon were more active at night than during day in all seasons but summer. The use of prepositioned arrays of acoustic receivers not only provides continuous data within a defined area, but provides insights into nocturnal behavior not previously examined.