In a search for enzymes involved in the formation of bile acids from 27-hydroxycholesterol in humans, the metabolism of this and other side-chain oxygenated steroids was studied in human liver microsomes and mitochondria. The microsomal fraction contained enzyme(s) catalyzing 7a-hydroxylation of 27-hydroxycholesterol and 3P-hydroxy-5-cholestenoic acid, whereas the 7a-hydroxylation of cholesterol and 3fl-hydroxy-5cholenoic acid was low. Only small amounts of 7phydroxylated products were formed. Purification and subfractionation of microsomal protein yielded a fraction of cytochrome P-450, which required NADPH and NADPH-cytochrome P-450 reductase and catalyzed 7a-hydroxylation of the side-chain oxygenated 3~-hydroxyd5-C,,-steroids but was inactive toward cholesterol. Added cholesterol did not inhibit the observed enzymatic activity. The results provide evidence that this enzyme is different from cholesterol 7a-hydroxylase. The mitochondrial fraction contained enzymeb) that catalyzed an isocitrate-dependent 7a- hydroxylation of 3fl-hydroxy-5-cholestenoic acid. The activity was much lower with 27-hydroxycholesterol. The mitochondrial fraction also catalyzed the oxidation of the 27-hydroxy group and contained a 38hydroxy-A5-steroid dehydrogenase active on 7a-hydroxylated C,,-steroids. The metabolic end product of the reactions catalyzed by these enzymes was 7ahydroxy-3-oxo-4-cholestenoic acid. A considerable fraction of the 7a-hydroxy-As intermediates was also converted to the corresponding 7p-hydroxysteroids, probably by way of the 7-oxosteroids, suggesting the presence of an epimerizing enzyme in the mitochondrial fraction. This study shows that 7a-hydroxy-3-oxo-4-cholestenoic acid, which is believed to be an important precursor of chenodeoxycholic acid in human beings, can be formed from 27-hydroxycholesterol or