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A rapid method was developed to measure cholesterol 7 α-hydroxylase activity of hepatic microsomes by the direct determination of the mass of 7 α-hydroxycholesterol formed. The method is based on the quantitative acetylation of the incubation mixture with [-3H]acetic anhydride and the separation of the biosynthetic 7 α-hydroxycholesterol as its diacetate by thin-layer chromatography on alumina. Amounts of 7 α-hydroxycholesterol as low as 0.1 nmole could be measured. A comparison of the proposed isotope derivative method with the previously used isotope incorporation method showed that the latter underestimated the enzyme activity by about 20 percent.