Female 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 0.1 N sodium 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 less than 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 less than 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 less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)