Bile from the toad, Bufo vulgaris formosus, was found to contain a number of minor bile acids along with two major bile acids, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-22-ene-24-carboxylic acid and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-23-en-26-oic acid. The following minor bile acids were identified by combined gas-liquid chromatography-mass spectrometry: cholic acid, allocholic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 alpha-cholest-22-ene-24-carboxylic acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 alpha-cholest-23-en-26-oic acid, varanic acid, and 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-24-methyl-5 beta-cholestan-26-oic acid. The fact that the toad bile contains not only cholic acid but also 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha, 12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acids, which have been recognized as biosynthetic intermediates of cholic acid in mammals, suggests that the toad is capable of synthesizing cholic acid by the same pathway as that for the biosynthesis of the C24 bile acid in mammals.