Abstract The gallbladder bile of patients with cholesterol gallstones is characterized by two abnormalities: (a) supersaturation with cholesterol and (b) accelerated nucleation of cholesterol monohydrate crystals. We studied the ability of purified human gallbladder mucin to nucleate artificial bile in vitro. Human gallbladder mucin at concentrations of 2 and 4 mg/ ml accelerated the nucleation time of cholesterol crystals in model bile. The mean number of cholesterol crystals in artificial bile incubated for 10 days with 4 mg/ml of human gallbladder mucin was 2327/ mm 3 (p < 0.01) vs. control of 51/mm 3. The number of crystals found in model bile was dependent on the concentration of human gallbladder mucin (2–16 mg/ml) and the time of incubation (4–14 days). Human gallbladder mucin was associated with an increase in the number of liquid crystals after 4 days of incubation, which then decreased in number as solid cholesterol monohydrate crystals formed. Nucleation by human gallbladder mucin was significantly increased only with cholesterol saturation indices >1.0, and in biles containing 10% but not 3% total lipid by weight. Pooled human gallbladder mucin from gallbladders with and without stones both increased nucleation significantly when compared with controls. Increased nucleation of saturated model bile was also observed with purified monkey cervical and bovine gallbladder mucin, but not with porcine gastric mucin. These observations provide further evidence that human gallbladder mucin may contribute to cholesterol gallstone formation in humans by accelerating nucleation of cholesterol monohydrate crystals from supersaturated gallbladder bile.