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 7 alpha-hydroxylation of 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, whereas the 7 alpha-hydroxylation of cholesterol and 3 beta-hydroxy-5-cholenoic acid was low. Only small amounts of 7 beta-hydroxylated 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 7 alpha-hydroxylation of the side-chain oxygenated 3 beta-hydroxy-delta 5-C27-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 7 alpha-hydroxylase. The mitochondrial fraction contained enzyme(s) that catalyzed an isocitrate-dependent 7 alpha-hydroxylation of 3 beta-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 3 beta-hydroxy-delta 5-steroid dehydrogenase active on 7 alpha-hydroxylated C27-steroids. The metabolic end product of the reactions catalyzed by these enzymes was 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. A considerable fraction of the 7 alpha-hydroxy-delta 5 intermediates was also converted to the corresponding 7 beta-hydroxysteroids, probably by way of the 7-oxosteroids, suggesting the presence of an epimerizing enzyme in the mitochondrial fraction.