BACKGROUND Nucleation from supersaturated bile of calcium salts of cholesterol and bilirubinate is essential in the formation of gallstone. Nucleation requires gallbladder mucin and its main component, glycoprotein, may contribute to gallstone formation by providing a nidus or matrix for precipitation of lipid components. However, biliary protein patterns of patients with gallstones have not been completely explored. METHODS We have tried to extract, isolate and characterize the proteins in patients with gallstones and without gallstones. 21 bile samples were obtained from patients with different types of gallstones and with no stones at cholecystectomy. Biliary protein concentrations were measured by Lowry and Bensadoun methods, and individual glycoproteins from each of the patients were compared by silver staining and densimetric quantification of Sodium Dodesyl Sulfate Polyacrylamide Gel Electrophoresis. RESULTS 1) Among 16 gallstones, 5 were cholesterol stones, 5 were calcium bilirubinate stones, and 6 were black pigment stones. 2) The mean protein concentration was highest in bile with cholesterol stones (47.6 mg/ml), 24.2 mg% in bile without gallstones, and 15.9 mg/ml in brown pigment stones. 3) Cholesterol gallstones were found to have 14.2 KD glycoproteins, whereas pigment stones were found to have 66 KD glycoproteins. CONCLUSIONS Gallbladder proteins from both cholesterol and pigment stones play an important role in the nucleation and growth of calcium salt crystals.