The effects of estradiol, testosterone, and pituitary extract on hepatic microsomal enzymes were studied in sham-operated and gonadectomized immature brook trout. Estradiol reduced the specific content of cytochromes P-450 and b5 by 70% or more in both groups. Testosterone and pituitary extract also decreased the levels of total P-450 and b5, but to a lesser extent. These latter effects were not evident when the contents of P-450 and b5 were normalized per g liver. Immunoblot analysis with antibodies to P-450 forms from a teleost (scup) showed the presence of cross-reacting proteins in control fish, presumed counterparts to the scup forms. Levels of a trout counterpart to P-450A (a putative testosterone 6 beta-hydroxylase) were strongly suppressed in estradiol-treated fish. A trout P-450B counterpart was suppressed in estradiol-treated fish, but less strongly than was the P-450A counterpart. The trout orthologue of hydrocarbon-inducible P-450E (P-450IA1), the aryl hydrocarbon hydroxylase (AHH) catalyst, was undetectable in any group, consistent with very low levels of AHH activity in these fish. Estradiol or pituitary extract also decreased the levels of NADH-cytochrome b5 and NADPH-cytochrome P-450 reductase activities in sham-operated but not in total P-450 or b5, suggesting an influence of gonads in maintaining normal levels of the reductases. The results support a prominent suppressive role for estrogens in producing the general sex differences in microsomal enzymes in fish liver and indicate that there could be affects on several P-450 forms. However, the mechanism of hormone action in this regulation is uncertain. The results also suggest the existence of unidentified, possibly gonadal, factors which contribute to the regulation of reductases in teleost liver.