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Methods were developed to determine the activity of the microsomal enzyme cholesterol 7α-hydroxylase in human liver. The enzyme assay could be performed with as little as 20 mg of fresh liver tissue, thus making the procedure applicable to specimens obtained by percutaneous liver biopsy. Optimal assay conditions were determined and the identity and radioactive purity of the reaction product, cholest-5-ene-3β,7α-diol (7α-hydroxycholesterol) were established. Specific enzyme activity was measured in a number of patients with disorders of lipid metabolism.