Nine rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) have been purified to electrophoretic homogeneity and assayed as potential catalysts of 15 beta-hydroxylation of 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate in a reconstituted system containing NADPH-cytochrome c reductase and dilauroylphosphatidylcholine. Of the nine isozymes, only cytochrome P-450i, which is present in adult female but not adult male rats, catalyzes the hydroxylation of 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. The reaction has an absolute requirement for cytochrome P-450i, NADPH-cytochrome c reductase, and NADPH, as well as a marked dependence on dilauroylphosphatidylcholine. Under optimal conditions, the Km,app was 69 microM, and the Vmax was 7.8 nmol min-1 nmol cytochrome P-450i-1. The affinity of cytochrome P-450i for the substrate, as expressed by the apparent spectral dissociation constant (Ks,app), was 20 microM. This female-specific isozyme had very low catalytic activity toward testosterone, and the metabolite profile from testosterone was distinct compared to the profiles obtained with the other eight isozymes. The results with androstane disulfate indicated that cytochrome P-450i is responsible for the sex-specific microsomal 15 beta-hydroxylase system in adult female rat liver, originally described by Gustafsson and Ingelman-Sundberg [(1974) J. Biol. Chem. 249: 1940-1945].