1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 α, 7 α, 12 α-trihydroxy-5 β-cholan-24-oate) and deoxycholate (3 α, 12 α-dihydroxy-5 β-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 α, 12 α, 23xi-trihydroxy-5 β-cholan-24-oic acid) and 3 α, 7 α, 12 α, 23 ξ-tetrahydroxy-cholanic acid (3 α, 7 α, 12 α, 23 ξ-tetrahydroxy-5 β-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed.
