Nine rat hepatic microsomal cytochromes P-450 ( P-450a- P-450i) have been purified to electrophoretic homogeneity and assayed as potential catalysts of 15β-hydroxylation of 5α-androstane-3α,17β-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 5α-androstane-3α,17β-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 K m,app was 69 μ m, and the V maxwas 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 ( K sapp ), was 20 μ m. 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 15β-hydroxylase system in adult female rat liver, originally described by Gustafsson and Ingelman-Sundberg [(1974) J. Biol. Chem. 249: 1940–1945].