The enzyme kinetics of estrogen 2-hydroxylase from rat liver microsomes has been investigated using two radiotracer methods. The conversion of [4-14C]estradiol to [4-14C]2-hydroxyestradiol by microsomes was examined by isolation of the catechol estrogen. [6,7-3H]2-Hydroxyestradiol was prepared and added at the end of the incubation as a recovery marker for quantitation. Initial velocity conditions of less than 10% product formation were established. The enzyme kinetics for estrogen 2-hydroxylase from male rat liver follows classical Michaelis-Menten kinetics producing a linear Lineweaver-Burk plot. The apparent Km for estradiol under these conditions was 2.2 microM; the Vmax for freshly prepared microsomes was 5.0 nmol/mg/min while that for microsomes stored at -70 degrees C was 0.51 nmol/mg/min. On the other hand, estrogen 2-hydroxylase from female rat liver microsomes did not follow Michaelis-Menten kinetics and yielded a nonlinear double reciprocal plot. Analogous kinetic data were obtained with another radiotracer assay measuring the release of 3H2O from [2-3H]estradiol. Thus, the estrogen 2-hydroxylases of male and female rat liver differ in enzyme kinetic properties. The upwardly curving Lineweaver-Burk plot of the kinetic data of the female rat liver microsomes suggests that more than one binding site for estradiol may exist on this enzyme complex and/or that this particular estrogen 2-hydroxylase system exhibits cooperative effects.