Stereospecific side-chain hydroxylations of 5β-cholestane-3α, 7α-diol were studied in mitochondrial and microsomal fractions of human liver. Incubation of 5β-cholestane-3α, 7α-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 5β-cholestane-3α, 7α,24-triols (24R and 24S) and 5β-cholestane-3α, 7α,26-triols (25R and 25S). A sensitive and reproducible radioactive assay to measure the formation of the different biosynthetic 5β-cholestanetriols was developed. Optimal assay conditions for human mitochondrial and microsomal systems were tentatively established.