(24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ. Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol. In the present investigation, we report formation of 7alpha, 24-dihydroxycholesterol in pig and human liver. 7alpha-hydroxylase activity toward both isomers of 24-hydroxycholesterol [(24S) and (24R)] was found in a partially purified and reconstituted cholesterol 7alpha-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes. In contrast, a purified enzyme fraction of pig liver oxysterol 7alpha-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol. 7alpha-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A. Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7alpha-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer. Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols. Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7alpha-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids.