HPLC determination of bilirubin conjugates in bile demonstrated a subgroup of patients with cholesterol gallstones who have more monoconjugated than diconjugated bilirubin in their gallbladder bile. None of the patients had abnormal liver function tests nor hemolysis. It was shown that this is not due to differences in liver secretion of the conjugates. In these patients, the more insoluble monoconjugated bilirubin might have provided the nucleus for the subsequent crystallization of cholesterol leading to the formation of cholesterol gallstones.