Guinea pig gallbladder bile contains chenodeoxycholic acid (62 +/- 5%), ursodeoxycholic acid (8 +/- 5%), and 7-ketolithocholic acid (30 +/- 5%). All three bile acids became labeled to the same specific activity within 30 min after [3H]cholesterol was injected into bile fistula guinea pigs. When a mixture of [3H]ursodeoxycholic acid and [14C]chenodeoxycholic acid was infused into another bile fistula guinea pig, little 3H could be detected in either chenodeoxycholic acid or 7-ketolithocholic acid. But, 14C was efficiently incorporated into ursodeoxycholic and 7-ketolithocholic acids. Monohydroxylated bile acids make up 51% and ursodeoxycholic acid 38% of fecal bile acids. After 3 weeks of antibiotic therapy, lithocholic acid was reduced to 6% of the total, but ursodeoxycholic acid (5-11%) and 7-ketolithocholic (15-21%) acid persisted in bile. Lathosterol constituted 19% of skin sterols and was detected in the feces of an antibiotic-fed animal. After one bile fistula guinea pig suffered a partial biliary obstruction, ursodeoxycholic and 7-ketolithocholic acids increased to 46% and 22% of total bile acids, respectively. These results demonstrate that chenodeoxycholic acid, ursodeoxycholic acid, and 7-ketolithocholic acid can all be made in the liver of the guinea pig.