The aim of this study was to determine the biliary pattern of conjugated bile acids after stimulation of their enterohepatic circulation. Conjugated bile acids were separated by reversed-phase ion-pair chromatography without prior derivatization. A MicroPak SP-C18-IP-4 column was used as non-polar matrix, and an ionic alkyl compound, tetrabutylammonium phosphate, was added to the mobile phase, which was a mixture of acetonitrile and water. Quantification was made by UV absorption at 210 nm with external standardization. In fourteen human patients with external biliary drainage after papillotomy there was preferential glycine conjugation. The mean values were 36.5% for glycocholic acid, 33% for glycochenodeoxycholic acid, and 10.0% for glycodeoxycholic acid. Only 15.2% of the biliary bile acids were taurine metabolites. Conjugates of ursodeoxycholic acid were below 2.1%. In most cases, conjugated lithocholic acid was not detected. Within 4 h after ingestion of a standardized meal there were no significant changes in the biliary bile acid pattern. In four dogs (beagles), glycine-conjugated bile acids were lacking. The mean values were 74.3% for taurocholic acid, 14.9% for taurodeoxycholic acid, and 5.3% for taurochenodeoxycholic acid. In six rabbits, 87.4% of biliary bile acids was identified as glycodeoxycholic acid and 5.3% as glycocholic acid. In conscious dogs, as well as in rabbits, the stimulation of biliary secretion by cholecystokinin and/or secretin had no effect on the biliary bile acid spectrum. Evidently, there is a difference in the biliary composition of conjugated bile acids between humans, dogs, and rabbits. Because of the different physicochemical behaviour of glycine- and taurine-conjugated bile salts, it seems difficult to compare the therapeutic effect of gallstone dissolution in various species.