A tritium release method is described for following the enzymic conversion of cholesterol to 7alpha-hydroxycholesterol. Incubations of rat liver subcellular preparations (containing microsomes) with [7alpha-3H]cholesterol or [7alpha,7beta-3H]cholesterol release the labeled hydrogen in the 7alpha position as 3H2O which, after counting, allows for the determination of the fraction of exogenous cholesterol converted to 7alpha-hydroxycholesterol. These findings document those recently reported by Van Cantfort, Renson, and Gielen (1975. Eur J. Biochem. 55:23). Analysis of incubation mixtures containing both [4-14C]cholesterol and either [7alpha-3H] or [7alpha,7beta-3H]cholesterol demonstrate that one atom of hydrogen (from the 7alpha position) is incorporated into H2O for every molecule of exogenous cholesterol that is converted to 7alpha-hydroxycholesterol. In the case of [7alpha-3H]cholesterol no label is retained by the product. With [7alpha,7beta-3H]cholesterol, one atom is released as 3H2O and one is retained by the product in the 7beta position. Microsomal incubations with [7alpha,7beta-3H]cholesterol were performed, followed by the acetylation of the steroid fractions with [14C]acetic anhydride. If intermixing of exogenous with endogenous cholesterol were complete during the enzymic reaction, one would expect the 3H: 14C ratio of the isolated cholesterol acetate to be four times that observed in the 7alpha-acetoxycholesterol acetate. Average values of 4.23 in one series and 4.03 in a second series indicate that intermixing was sufficiently complete to use the tritium release method as an indicator of mass conversion.