Estradiol (E2) metabolites formed in vitro by microsomes from the marine teleosts winter flounder (Pseudopleuronectes americanus) and scup (Stenotomus chrysops) included at least seven products detected by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The least polar metabolite was shown to be estrone by chromatographic and mass spectrometric identity with authentic estrone. Chromatographic analyses coupled with dual-label experiments also indicated formation of the catecholestrogen 2-hydroxyestradiol (2-OH-E2), which was the most prominent metabolite determined by TLC. Analysis of microsomal E2 2-hydroxylase activity by measuring the specific release of 3H2O from [2-3H]E2 indicated that it is mediated by cytochrome P-450. E2 2-hydroxylase activity normalized to microsomal protein was lower in females than in males for microsomes from both mature scup and winter flounder. Activity normalized to liver weight or body weight in female winter flounder was also lower than that in males. However, activity normalized to cytochrome P-450 content did not show sex differences in either species. E2 2-hydroxylase activity per nanomole cytochrome P-450 was reduced in scup treated with beta-naphthoflavone, which induces the hydrocarbon hydroxylase cytochrome P-450E. Studies employing reconstituted P-450E and microsomes preincubated with polyclonal antibodies against P-450E confirmed that this isozyme does not catalyze E2 2-hydroxylase activity in microsomes. However, preliminary work with scup cytochrome P-450A suggests that it may be an E2 2-hydroxylase. The studies establish that catecholestrogen formation is prominent in fish liver and that it is sexually differentiated, but further investigation is required to define the catalysts as well as the significance and regulation of this function.