Previously, it has been demonstrated that nonylphenol (NP) has a dual function in regulating reproductive hormones by: (1) increasing the activity of steroid metabolizing enzymes at low concentration, that does not induce vitellogenin (Vtg) and zona radiata proteins and (2) decreasing the activity of these enzymes at higher concentration [Environ. Health Perspect. 105 (1997) 109; Environ. Toxicol Chem. 16 (1997) 2576]. To more precisely understand the estrogenic effects of NP in fish and a possible interference with steroid hormone metabolism, we investigated in the Atlantic salmon the identity of the cytochrome P450 enzymes involved in NP hydroxylation. Up to 9 metabolites were separated by radio-HPLC when [R-(14)C]-4n-NP was incubated with hepatic microsomes from juvenile salmon. In control fish the major metabolites were identified as monohydroxylated products at omega-, (omega-1)- and (omega-2)-positions of the alkyl chain of 4n-NP. Salmon hepatic microsomes formed omega-, (omega-1)- and (omega-2)-lauric acid (LA) hydroxylation products. The potency of alpha-naphthoflavone, ketoconazole and ethynylestradiol (non-specific, but strong inhibitors of CYP1A, 2K and 3A, respectively) on LA and NP hydroxylations were assessed in the present study. Ketoconazole inhibited omega-, (omega-1)- and (omega-2)-hydroxylations of LA and 4n-NP and was the only inhibitor of omega-hydroxylation of both substrates. Ethynylestradiol specifically inhibited (omega-1)- and (omega-2)-hydroxylations of LA as well as 4n-NP. Interestingly, the lowest NP dose (1 mg/kg) was the most potent inducer of NP-metabolites formation. These results suggest the involvement of CYP2M- and 2K-like enzymes in terminal and subterminal hydroxylations of 4n-NP respectively, and was confirmed by the competitive inhibition between LA and 4n-NP. The production of one unidentified 4n-NP metabolite was not affected by any of the chemicals used, suggesting a possible ring hydroxylation with involvement of another cytochrome P450 isoform. Our data reveal a novel aspect of CYP isozymes involvement in NP metabolism that may complicate the assessment of its endocrine effects. Hence, the regio-selective hydroxylation of endocrine disruptors, such as NP, by CYP isozymes is revealed as a possible new marker of estrogenicity.