Spotted seatrout, Cynoscion nebulosus, spawning locations as well as associated environmental variables were determined for Tampa Bay, Florida during the 2004 spawning season using a mobile hydrophone survey. Hydrophones, a type of underwater microphone, can be used to detect and record spawning sounds of soniferous fishes. During their spawning season in Tampa Bay which generally occurs between March and September, mature male spotted seatrout generate sounds associated with courtship in the crepuscular and evening periods by vibrating sonic muscles against the swim bladder. Active spawning sites can be located using hydrophones to find these calling males. Using a random stratified sampling method, 760 stations within Tampa Bay (46 % of the sampling universe) were sampled over the 2004 spawning season. Only 8% of sampled stations had large aggregations of spotted seatrout. Spawning, determined by the sound produced by large aggregations, was detected throughout the bay except for Hillsborough Bay and was most common in the lower bay and eastern region of the middle bay. Presence of submerged aquatic vegetation (SAV), proximity to shoreline, as well as high dissolved oxygen values and shallow depth were positively correlated with spawning areas. Courtship calls of sand seatrout, Cynoscion arenarius, and silver perch, Bairdiella chrysoura were also detected during the survey as they share an overlapping spawning season with spotted seatrout. Aggregations of all three species rarely occurred simultaneously. Sand seatrout and silver perch used different habitats within Tampa Bay to spawn and spawned with a much greater frequency than spotted seatrout. Courtship calls of spotted seatrout were analyzed both by ear and by received sound level to determine if signal processing could be used to assess courtship sound recordings. However, there was no clear relationship between the two methods.