The mechanism of aromatic hydroxylation of estrogens by cytochrome P450 enzymes has been examined by comparing the oxidation of estrone with that of substrates carrying additional aromaticity such as equilenin and the structural analog 2-naphthol. Hamster liver microsomes preferentially catalyzed the conversion of estrone to 2-hydroxyestrone (Km = 30 and 25 microM and Vmax = 1497 and 900 pmol (mg of protein)-1 min-1 for 2- and 4-hydroxyestrone formation, respectively). In contrast, equilenin was hydroxylated exclusively at C-4 of the steroid ring system and 2-naphthol at the corresponding C-1 position (Km = 67 and 42 microM and Vmax = 2083 and 3226 pmol (mg of protein)-1 min-1 for 4-hydroxyequilenin and 1,2-dihydroxynaphthalene formation, respectively). This shift in the specificity of hydroxylation was due to the introduction of additional aromaticity at ring B of equilenin, because hamster liver microsomes are known not to contain any estrogen-4-hydroxylase, only estrogen-2-hydroxylase activity catalyzed by cytochrome P450 3A family enzymes. The exclusive 4-hydroxylation of equilenin is proposed to be due to a preferred delocalization of the naphthoxy radical an intermediate in the hydroxylation, to C-4, whereas delocalization to C-2 requires additional activation energy and is energetically not favored. Based on these electronic considerations, a mechanism of aromatic hydroxylation of estrogens is proposed which features hydrogen abstraction from the phenolic hydroxy group, electron delocalization of the phenoxy radical to a carbon-centered radical, and subsequent formation of catechol metabolites by hydroxy radical addition at C-2 or C-4 depending on steric or electronic constraints.