The known activity of cytochrome P450 46A1 (P450 46A1) is 24(S)-hydroxylation of cholesterol. This reaction produces biologically active oxysterol, 24(S)-hydroxycholesterol, and is also the first step in enzymatic degradation of cholesterol in the brain. We report here that P450 46A1 can further metabolize 24(S)-hydroxycholesterol, giving 24,25- and 24,27-dihydroxycholesterols in both the cell cultures transfected with P450 46A1 cDNA and the in vitro reconstituted system with recombinant enzyme. In addition, P450 46A1 was able to carry out side chain hydroxylations of two endogenous C27-steroids with and without a double bond between C5-C6 (7alpha-hydroxycholesterol and cholestanol, respectively) and introduce a hydroxyl group on the steroid nucleus of the C21-steroid hormones with the C4-C5 double bond (progesterone and testosterone). Also, P450 46A1 was found to metabolize xenobiotics carrying out dextromethorphan O- and N-demethylations, diclofenac 4'-hydroxylation, and phenacetin O-deethylation. Thus, substrate specificities of P450 46A1 are not limited to cholesterol and include a number of structurally diverse compounds. Activities of P450 46A1 suggest that, in addition to the involvement in cholesterol homeostasis in the brain, this enzyme may participate in metabolism of neurosteroids and drugs that can cross the blood-brain barrier and are targeted to the central nervous system.