AbstractFemale Hartley guinea pigs fed a 0.5% cholesterol‐supplemented diet were found to form pigmented gallstones after 6 weeks (17/23) and 12 weeks (11/11), while only 2 of 44 animals fed a trace cholesterol diet formed gallstones over a comparable period. The light brown stones consisted primarily of aggregates of fine granular crystals, morphologically similar to calcium bilirubinate crystals. The stones were soluble in 0.1Nsodium hydroxide and were found to contain a substance which co‐migrated with unconjugated bilirubin during thin‐layer chromatography.Despite hypercholesterolemia (202 ± 34 vs. 59 ± 22 mg per dl in controls, p<0.05) and fatty infiltration of the liver, cholesterol‐fed animals had a lithogenic index of only 0.22 ± 0.04 in gallbladder bile as compared to a lithogenic index of 0.02 ± 0.01 in animals fed the trace cholesterol diet. Accordingly, no cholesterol monohydrate crystals were found in any animals. Hematocrits among cholesterol‐fed animals (47.6 ± 1.2%) were lower than those of controls (54.8 ± 1.3%, p<0.05) probably as a result of the cholesterol‐induced hemolytic anemia which has been reported by others in this species.Fasting gallbladder volume was greater in cholesterol‐fed animals (2.4 ± 0.18 ml) than in controls (1.7 ± 0.11, p<0.0025), and a comparable increase in gallbladder dry tissue mass was found. There was no evidence of biliary obstruction, however, and the gallbladder contractile response to octapeptide cholecystokinin was comparable in both groups. The increase in gallbladder volume appeared to result from: (a) an increase in hepatic bile salt secretion with an associated increase in bile flow and (b) a decrease in the capacity of the gallbladder to absorb fluid from hepatic bile (6.0 ± 1.4 mg fluid per hr per mg tissue with cholesterol diet vs. 12.9 ± 0.9 in controls, p<0.0005). Total bile salt concentration was also decreased in gallbladder bile of cholesterol‐fed animals (13.79 ± 0.51 vs. 18.56 ± 2.19 mmoles per liter in controls, p<0.05), probably as a result of increased hepatic bile flow and decreased gallbladder absorption. The absence of cholesterol gallstones in this model is consistent with the failure to supersaturate bile with cholesterol. The reasons for pigment gallstone formation are less clear but their occurrence may be the result of a cholester