We recently reported that nb/nb mice with hereditary hemolytic anemia spontaneously developed calcium bilirubinate gallstones. We undertook this study to determine whether the differences in gallbladder bile composition were due to altered gallbladder function or hepatic bile composition. Hepatic bile was obtained by cholecystostomy after common bile duct ligation. We found that (a) the hepatic bile of nb/nb mice with or without stones had higher concentrations of unconjugated (p less than 0.001) and total bilirubin (p less than 0.001) but lower concentrations of bile acids (p less than 0.05) than that of control mice; (b) the concentrations of total calcium and hydrogen ion were similar in all groups; (c) nb/nb mice with stones compared with nb/nb mice without stones had higher concentrations of unconjugated (p less than 0.05) and total bilirubin (p less than 0.05); (d) the outputs of unconjugated and total bilirubin of nb/nb mice with or without stones were higher than control mice (p less than 0.001) while bile acid, hydrogen ion, and calcium outputs were similar in all groups; (e) nb/nb mice with stones had higher outputs of unconjugated (p less than 0.005) and total bilirubin (p less than 0.05) than nb/nb mice without stones; (f) in nb/nb mice with stones, but not in those without stones or control mice, unconjugated bilirubin output was associated with bile acid output (p less than 0.001); and (g) unconjugated bilirubin and total bilirubin outputs were significantly correlated in all groups (p less than 0.001). Thus, an increased concentration and amount of unconjugated bilirubin in nb/nb hepatic bile is an essential factor in hemolysis-induced gallstone formation and modification of this abnormal nb/nb hepatic bile within the gallbladder promotes stone formation.