Hepatic mitochondrial cytochrome P-450 has been partially purified from the non-induced rat liver. The purification consisted of solubilization with cholate, polyethylene glycol fractionation, chromatographic separation using omega-amino-n-octyl Sepharose 4B column, and chromatography on hydroxylapatite. The overall purification of the enzyme from the solubilized extract was about 22-fold on the basis of specific content of cytochrome P-450, and 50-fold on the basis of specific activity. The partially purified enzyme was active for both 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and 5 beta-cholestane-3 alpha, 7 alpha-diol. That the enzyme activities for these substrates were not due to two different enzymes but to the same active site of a single enzyme protein was shown by several pieces of evidence, i.e., behavior to thermal inactivation, pH-dependency of the reaction velocities, experiments with mixed substrates, and behavior towards inhibitors and activators. The lower Km value and the higher Vmax for 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol compared to 5 beta-cholestane-3 alpha, 7 alpha-diol seem to be important factors for the regulation mechanism that keeps the ratio of cholic acid/chenodeoxycholic acid constant in rat bile.