Adrenocorticotrophic hormone (ACTH), angiotensin II (AII), and the urophysial peptides, urotensins I and II (UI and UII), stimulate cortisol secretion by interrenal preparations of seawater (SW) and freshwater (FW) adapted trout. Steroid secretion was not disturbed in sham-treated control groups. The increased cortisol secretion following perifusion of tissue with 10(-7) M ACTH in combination with 10(-7) M AII, 10(-7) M UI, or 10(-7) M UII was greater than after separate administration of ACTH, AII, UI, or UII. These responses were no greater than the summation of the separate effects of ACTH, AII, or UII in SW and FW derived tissue or of ACTH and UI in FW derived tissue. However, the increased cortisol secretion (600-700%) after UI and ACTH in combination in SW adapted fish was significantly higher than the summated responses (100-200%) to UI and ACTH when administered separately. These results suggest that in SW fish interrenal UI enhances the steroidogenic action of ACTH, a potentially important response in SW teleost fish.