This study was performed to determine whether biliary cholesterol nucleates primarily from vesicles or micelles. Twenty gallbladder biles and 12 hepatic biles from patients with gallstones as well as 16 model biles were examined. The nucleation times (days) of the biles as well as their isolated vesicular and micellar fractions were determined and their lipid composition was analyzed. In 41 of 46 comparisons, cholesterol nucleated faster from vesicles than micelles; in only one case was the opposite found. The mean (± S.D.) nucleation times of vesicles and micelles in gallbladder biles were 8.9 ± 5.4 vs. 15.4 ± 8.6, in hepatic biles 14.6 ± 9.4 vs. 20.6 ± 9.1, and in model biles 9.0 ± 3.7 vs. 18.9 ± 9.1 days, respectively. All these differences were significant (p < 0.005). Gallbladder biles (n = 7) devoid of vesicles nucleated more slowly (9.0 ± 9.5 days), as compared to gallbladder biles (n = 13) containing vesicles (3.8 ± 2.2 days). The nucleation time of gallbladder and hepatic biles was significantly correlated with the nucleation time of the vesicles from these biles (r = 0.847, p < 0.05). There was no correlation with the nucleation time of micelles from the same biles. The percentage of cholesterol carried by vesicles in bile was positively correlated to the molar percentage of biliary cholesterol and the cholesterol saturation index and negatively correlated to the molar percentage of bile salts. Our data suggest that phospholipid vesicles are the major vehicle for cholesterol precipitation in bile as well as an important determinant of the nucleation time of bile.