The inhibition of estrogen 2-hydroxylase by androgens was demonstrated in screening assays and has been further investigated under initial velocity conditions. The ability of testosterone, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone to block the conversion of estradiol to 2-hydroxyestradiol by male rat liver microsomal preparations was determined using two radiotracer methods--the conversion of [4-14C]estradiol to [4-14C]2-hydroxyestradiol and the release of 3H2O from [2-3H]estradiol. The apparent Ki's for the androgens ranged from 12.0 to 14.0 microM, with the apparent Km for the substrate estradiol in these assays of 2.08 microM. Multiple inhibition studies with the androgens and 2-bromoestradiol, an effective estrogen inhibitor, in male rat liver microsomes resulted in Dixon plots consisting of a series of nonparallel, intersecting lines. Thus, the androgens and 2-bromoestradiol are non-exclusive inhibitors, i.e. the binding of one compound to the enzyme does not interfere with the binding of the other. These interactions of androgens suggest that the steroid hormonal environment be considered in the examination of the physiological role(s) of the estrogen 2-hydroxylase and the catechol estrogen products.