Physiological levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17, 20-P) stimulated time- and dose-dependent increases in cortisol production by rainbow trout (Oncorhynchus mykiss) interrenal tissue cultured in vitro. Significant stimulation occurred in response to 100, 300, and 1000 ng/ml of 17,20-P. Lower doses were ineffective. Elevated cortisol levels were observed 1 hr after addition of 300 ng/ml 17,20-P. No additive or synergistic interaction was evident between human adrenocorticotropin fragment 1-24 (ACTH1-24) and 17, 20-P in stimulating cortisol secretion, although 300 ng/ml 17,20-P could further enhance cortisol production above levels already stimulated by 300 ng/ml ACTH. 17alpha, 20alpha-dihydroxy-4-pregnen-3-one also stimulated cortisol secretion, but was only half as effective as 17,20-P. Estradiol-17beta, testosterone, and 11-ketotestosterone had no effect on cortisol secretion. Inhibitors of mRNA and protein synthesis had no effect on 17,20-P-stimulated cortisol production. Radiotracer studies demonstrated that the bioconversion of 17,20-P to cortisol could fully account for the cortisol produced by the interrenal in response to 17,20-P and demonstrated that rainbow trout interrenal cells contain an active 20beta-hydroxysteroid dehydrogenase. These data suggest that 17,20-P may be a regulator of cortisol production during the periovulatory period in salmonid fishes.