Abstract:The bile acid composition of bile obtained from the hepatopancreatic ducts of three species of sperm whales (Cetacea: Physeteridae) was investigated. Bile acids were isolated by adsorption chromatography and analyzed by sequential HPLC, SIMS, and GLC‐MS. In each species the dominant bile acids were deoxycholic acid (a secondary bile acid formed by bacterial 7α‐dehydroxylation of cholic acid), and chenodeoxycholic acid (a primary bile acid) which together composed more than 86% of biliary bile acids in all three species. InPhyseter catodon(sperm whale) deoxycholic acid constituted 79%, and inKogia breviceps(pygmy sperm whale) it was 61% of biliary bile acids. The sperm whale, which differs from other whales in having a remnant of a large intestine, is the second mammal identified to date in which deoxycholic acid is the predominant bile acid. The high proportion of deoxycholic acid indicates that in the Physeteridae, anaerobic fermentation occurs in its cecum, and that bile acids undergo enterohepatic cycling. Also found were minor proportions of cholic acid, as well as bacterial derivatives of chenodeoxycholic acid (ursodeoxycholic acid, lithocholic acid, and the 12β‐epimer of allo‐deoxycholic acid). Bile acids were conjugated with taurine in all species; however, in the sperm whale (Physeter) glycine conjugates were present in trace proportions. The bile acid hydroxylation pattern (12α‐ but not 6α‐hydroxylation), lack of primary 5α‐ (allo) bile acids, and presence of glycine conjugated bile acids suggests the possibility that sperm whales originated from an