Human liver microsomes catalyze an efficient 25-hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degradation in the biosynthesis of cholic acid. The enzyme responsible for the microsomal 25-hydroxylation has been unidentified. In the present study, recombinant expressed human P-450 enzymes have been used to screen for 25-hydroxylase activity towards 5beta-cholestane-3alpha, 7alpha,12alpha-triol. High activity was found with CYP3A4, but also with CYP3A5 and to a minor extent with CYP2C19 and CYP2B6. Small amounts of 23- and 24-hydroxylated products were also formed by CYP3A4. The Vmax for 25-hydroxylation by CYP3A4 and CYP3A5 was 16 and 4.5 nmol/(nmolxmin), respectively. The Km was 6 microM for CYP3A4 and 32 microM for CYP3A5. Cytochrome b5 increased the hydroxylase activities. Human liver microsomes from ten different donors, in which different P-450 marker activities had been determined, were incubated with 5beta-cholestane-3alpha,7alpha, 12alpha-triol. A strong correlation was observed between formation of 25-hydroxylated 5beta-cholestane-3alpha,7alpha,12alpha-triol and CYP3A levels (r2=0.96). No correlation was observed with the levels of CYP2C19. Troleandomycin, a specific inhibitor of CYP3A4 and 3A5, inhibited the 25-hydroxylase activity of pooled human liver microsomes by more than 90% at 50 microM. Tranylcypromine, an inhibitor of CYP2C19, had very little effect on the conversion. From these results, it can be concluded that CYP3A4 is the predominant enzyme responsible for 25-hydroxylation of 5beta-cholestane-3alpha, 7alpha,12alpha-triol in human liver microsomes.