Mono-, di-, tri-, and tetrahydroxy-5β-cholestan-26-oic acids were incubated with rat liver homogenate (800 g supernatant and light mitochondrial fraction) to study substrate specificity in the side-chain cleavage reaction (β-oxidation) of bile acid biosynthesis. The C 2 7 -intermediates (5β-cholest-24-en-26-oic acids and 24-hydroxy-5β-cholestan-26-oic acids) in β-oxidation and the corresponding C 2 4 -bile acids were quantitatively determined by capillary gas chromatography. Monohydroxy-5β-cholestan-26-oic acid was not converted into C 2 4 -bile acid. Di- and trihydroxy-5β-cholestan-26-oic acids were effectively transformed into the C 2 7 -intermediates and C 2 4 -bile acids. Tetrahydroxy-5β-cholestan-26-oic acids were also converted into C 2 7 -intermediates and corresponding C 2 4 -bile acids. The intermediate 24-hydroxy-5β-cholestan-26-oic acids could not be detected in the products by incubation with the light mitochondrial fraction. The total specific activity of protein in the light mitochondrial fraction for the production of C 2 7 -intermediates and C 2 4 -bile acids was higher than that of 800 g supernatant solution. The effects of the number and the position of hydroxyl groups on the side-chain degradation are discussed.