The role of bilirubin conjugates in the formation of pigment gallstones is not known. In this study, we completely solubilized and then analyzed by high-performance liquid chromatography specimens of black pigment gallstones from eight nb/nb mice with hereditary hemolytic anemia. Each dried gallstone specimen of about 200 μg was dissolved in 5 ml of dimethyl sulfoxide/0.15 M HCl/50 mM disodium-EDTA (8:1:1 by volume) at room temperature. Stone dissolution was complete by 30 min as monitored by the A456 and direct observation, and no oxidative products of bilirubin were observed in the visible spectrum, 350 to 750 nm. By high-performance liquid chromatography, the intact tetrapyrroles were separated as diconjugated and monoconjugated bilirubins; unconjugated bilirubin was resolved as XIII, IX and III α-isomers. The isocratic solvent system used was 0.1 M di-n-dodecylamine acetate/0.1 M di-n-octylamine acetate (4:1, v/v) in methanol, pH 7.4, at a flow of 1 ml per min. Diconjugated bilirubin accounted for 6.0 ± 2.4 molar % (mean ± S.E.), monoconjugated bilirubin for 37.4 ± 8.4% and unconjugated bilirubin for 56.3 ± 8.9% of the solubilized pigments. The IX α-isomer represented 96 ± 1.9% of the unconjugated bilirubin. The presence of bilirubin conjugates in gallstones was confirmed by ethylanthranilate diazotization: the conjugated azodipyrrole in stone had the same retention time as that of conjugated azodipyrrole from rat and mouse bile. A majority of the bilirubin conjugates was sensitive to β-glucuronidase of liver origin, indicating that the C-1 glucuronide ester was present. We conclude that (i) monoconjugated bilirubins are a significant component of black pigment gallstones from mice with hereditary hemolytic anemia, and (ii) the IX α-isomer is the predominant unconjugated bilirubin isomer in these gallstones. Monoconjugated bilirubin may play a role in black pigment stone formation as a coprecipitant and source of unconjugated bilirubin.