Urotensin I (UI), one of the biologically active peptides isolated from the caudal spinal cord and urophysis of fish, exhibits a strong sequence homology with mammalian corticotropin-releasing factor (CRF). We have applied an antiserum to ovine CRF that cross-reacts completely with UI to sections taken from the caudal spinal cord and urophysis of the channel catfish (Ictalurus punctatus). Using the indirect immunofluorescence technique, the presence of striking Ul-like immunoreactivity was observed within discrete neuronal structures. UI immunoreactivity was found within large-diameter neuronal perikarya in spinal cord segments immediately rostral to the urophysis, in fascicles of nerve fibers as the urophysis emerges, and in a dense plexus of nerve fibers and terminals which abut capillary loops within the urophysis. The localization of UI immunoreactivity within discrete neurosecretory neurons of this system will make it possible to more fully determine the nature of the regulatory mechanisms controlling its secretion.