The effects of a standard rodent chow were compared with those of a semisynthetic diet of known composition (with and without added cholesterol) in the prairie dog model of cholesterol cholelithiasis. Gallstone incidence was 40% higher in animals fed a semisynthetic diet plus cholesterol compared to chow plus cholesterol. The semisynthetic diet plus cholesterol caused significant increases in tissue cholesterol levels (serum, liver and bile) and lithogenic index, but significant decreases in the activity of hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and cholesterol 7α-hydroxylase compared to chow plus cholesterol. Histologic study of liver sections revealed that the semisynthetic diet plus cholesterol resulted in moderate to marked portal tract changes characterized by bile duct proliferation, inflammatory infiltration and fibrosis, whereas the cholesterol-supplemented chow diet caused only slight bile duct proliferation with minimal inflammation and fibrosis in the portal areas. Dietary hyodeoxycholic acid prevented cholesterol gallstones and biliary cholesterol crystals when added to either chow plus cholesterol or semisynthetic plus cholesterol diets. The hyodeoxycholic acid supplements also prevented the development of severe histopathologic alterations along the portal tracts. Biliary cholesterol levels were elevated in prairie dogs fed cholesterol plus hyodeoxycholic acid; these animals had liquid crystals in the bile, and hyodeoxycholic acid and its 6β-isomer became the major biliary bile acids. A semisynthetic diet plus cholesterol is superior to a high cholesterol chow diet for gallstone formation and prevention studies, but in prolonged feeding experiments, the potential hepatotoxicity of this diet in the prairie dog must be appreciated.