The inhibition of estrogen 2-hydroxylase by 2-methoxyestrogens was demonstrated in screening assays and has been further investigated under initial velocity conditions. The ability of 2-methoxyestradiol and 2-methoxyestrone to block the conversion of estradiol to 2-hydroxyestradiol by male rat liver microsomal preparations was determined by measuring the release of 3H2O from [2-3H]estradiol. The apparent Kis were found to be 34.86 microM for 2-methoxyestradiol and 18.65 microM for the methoxyestrone, with the apparent Km for the substrate estradiol in these essays of 3.21 microM. Mixed inhibition studies with the methoxyestrogens and 2,4-dibromoestradiol, an effective estrogen 2-hydroxylase inhibitor, in male rat liver microsomes resulted in Dixon plots consisting of a series of parallel lines. Thus, methoxyestrogens and 2,4-dibromoestradiol are mutually exclusive inhibitors, i.e., the binding of one compound to the enzyme interferes with the binding of the other. These results indicate that the compounds are interacting at the same enzymatic site. Finally, a method utilized to measure estrogen 2-hydroxylase activity in vitro is a radioenzymatic assay involving addition of catechol o-methyltransferase (COMT) and radiolabeled S-adenosylmethionine, and the amount of catechol estrogens formed is determined by the amount of radiolabeled methoxyestrogens isolated. The results described here demonstrate inhibition of estrogen 2-hydroxylase by methoxyestrogens; however, under enzymatic conditions of low product formation, the estrogen 2-hydroxylase inhibitory effect of catechol estrogen products from the radioenzymatic assay would be insignificant. Thus, these interactions of methoxyestrogens suggest that the steroid hormonal environment be considered in the examination of estrogen 2-hydroxylase and the catechol estrogen products. off