Stereospecific side-chain hydroxylations of 5beta-cholestane-3alpha, 7alpha-diol were studied in mitochondrial and microsomal fractions of human liver. Incubation of 5beta-cholestane-3alpha, 7alpha-diol resulted in hydroxylations at C-12, C-24, C-25, and C-26. Hydroxylations at C-24 and C-26 were accompanied by the introduction of additional asymmetric carbon atoms at C-24 and C-25 respectively, that led to the formation of two distinct pairs of diastereoisomers, namely 5beta-cholestane-3alpha, 7alpha,24-triols (24R and 24S) and 5beta-cholestane-3alpha, 7alpha,26-triols (25R and 25S). A sensitive and reproducible radioactive assay to measure the formation of the different biosynthetic 5beta-cholestanetriols was developed. Optimal assay conditions for human mitochondrial and microsomal systems were tentatively established.The mitochondrial fraction was found to predominantly catalyze the 26-hydroxylation of 5beta-cholestane-3alpha, 7alpha-diol with the formation of the 25R-diastereoisomer of 5beta-cholestane-3alpha, 7alpha,26-triol as the major product. In the microsomal fraction, on the other hand, 25-hydroxylation was more efficient than 26-hydroxylation and accounted for 6.4% of the total hydroxylations. The microsomes catalyzed the formation of both diastereoisomers of 5beta-cholestane-3alpha, 7alpha,26-triol (25R and 25S, 4.2 and 1.6% respectively). These experiments suggest that the initial step in the degradation of the steroid side chain during the biosynthesis of chenodeoxycholic acid in man is mediated by the mitochondria, and involves the formation of the 25R-diastereoisomer of 5beta-cholestane-3alpha, 7alpha,26-triol. The role of the microsomal 25- and 26-hydroxylated intermediates requires further exploration.