To elucidate the importance and the changing patterns of biliary concanavalin A-binding proteins (CPs) in the early stage of cholesterol gallstone formation. CP concentration and nucleation activity were measured by lectin affinity chromatography in bile samples of patients with cholesterol gallstones, pigment gallstones, gallbladder cholesterosis and non-biliary diseases. The concentrations of CPs were much higher in patients with cholesterol gallstones (0.39 ± 0.11 g/L, n = 36, P < 0.01) or gallbladder cholesterosis (0.40 ± 0.09 g/L, n = 9, P < 0.01) than in those with pigment gallstones (0.2 ± 0.12 g/L, n = 7) and/or non-biliary diseases (0.27 ± 0.09 g/L, n = 10). Pronucleating activities were much stronger in patients with cholesterol gallstones (nucleation time ratio: 0.57 ± 0.21, n = 5, P < 0.01 vs pigment gallstones and/or non biliary diseases) and gallbladder cholesterosis (nucleation time ratio: 0.44 ± 0.23, n = 5, P < 0.01 vs pigment gallstones or non-biliary diseases). The binding percentages of CPs to model biliary vesicles were also higher for patients with cholesterol gallstones (n = 6) than those with pigment gallstones (n = 6) (2.4% ± 0.9% vs 0.9% ± 0.5%, P < 0.01). Hypersecretion of CPs, especially those in vesicular phase, may be an important change in the early stage of cholesterol gallstone formation.