Epidemiological studies have provided conflicting information about the relationship between fat consumption and gallstone formation. We studied cholesterol gallstone formation in prairie dogs after 1 wk of the following diets: (group A) a control diet with no added cholesterol and 5% of calories from corn oil, (group B) 1.2% cholesterol with 5% of calories from corn oil or (group C) 1.2% cholesterol with 40% of calories from corn oil. In controls serum cholesterol was 58.9 +/- 4.5 mg/dl, gallbladder bile was unsaturated with cholesterol (cholesterol saturation index = 0.7 +/- 0.1; cholesterol = 3.8 mmol/L) and none of 12 animals formed cholesterol crystals or stones. The low-fat diet supplemented with cholesterol (group B) increased serum and biliary cholesterol concentrations to 292 +/- 76 mg/dl and 7.5 +/- 1.1 mmol/L, respectively (p < 0.05), but cholesterol saturation index was only modestly increased (1.1 +/- 0.1) and in only one of eight animals did cholesterol monohydrate crystals develop. Group C, animals, which received cholesterol plus high levels of corn oil, had higher serum cholesterol levels (457 +/- 66 mg/dl), higher biliary cholesterol concentrations (16.6 +/- 1.3 mmol/L), higher cholesterol saturation indexes (1.7 +/- 0.1) and increased incidence of cholesterol gallstones (5 of 11). The two cholesterol-supplemented diets increased biliary phospholipid concentrations, decreased the ratio of cholic/chenodeoxycholic acid and increased the proportion of biliary lecithins containing linoleic acid, but these abnormalities were greatest in group C, which was given large amounts of corn oil. These findings suggest that cholesterol gallstone formation in the prairie dog is accelerated by increased dietary omega-6 polyunsaturated triglycerides.