One of each constitutive form of cytochrome P-450 from liver microsomes of adult male and female rats was purified essentially following the same method to an apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights estimated by the electrophoresis were 52,000 and 50,000 for forms of cytochrome P-450, P-450-male, and P-450-female, purified from male and female rats, respectively. In addition, the purified preparations of P-450-male and P-450-female showed properties different from each other with respect to spectral characteristics and catalytic activities. In Ouchterlony double diffusion plates, partially purified rabbit immunoglobulin G (IgG) raised against P-450-male and P-450-female showed very weak or no cross-reactivity with P-450-female and P-450-male, respectively. From these results, P-450-male was confirmed to be a form distinct from P-450-female. The anti-P-450-male and anti-P-450-female antibodies, which had been further purified by immunoadsorption, did not form any apparent precipitation bands with liver microsomes from untreated female and male rats, respectively. Supporting this, radial immunodiffusion analysis for P-450-male and P-450-female with an agarose gel impregnated with the rabbit antibodies showed that P-450-male and P-450-female appear in liver microsomes rather specifically depending on the sex hormones. Based on these results, sex differences in drug metabolism in the rat were confirmed as explicable, at least in part, by the presence of distinct forms of cytochrome P-450 in microsomes of male and female rats.