The amount and composition of gallbladder bile acids of eight germfree and four conventional pure-bred Beagle dogs were determined. Unlike in other mammalian species, no major differences in bile acid composition were found between germfree and conventional dogs. Germfree dog bile contained on the average 94.9% cholic acid and 4.6% chenodeoxycholic acid. Conventional dog bile contained 83.6% cholic acid, 3.7% chenodeoxycholic acid and in addition, 12.1% deoxycholic acid. Bile from both germfree and conventional dogs contained traces of keto-bile acids. The average amounts of total bile acids in mg/ml bile were 36.8 ± 2.6 (germfree) and 28.5 ± 2.9 (conventional). Except for differences in conjugation, dog and human biliary bile acid patterns are qualitatively comparable, and differ from those in rats and mice in that muricholic and related bile acids are found only in the latter group.