Aggregation of cholesterol-phospholipid vesicles in supersaturated biles precedes cholesterol crystal formation. In this study we examined the relationship between the percentage of cholesterol carried by vesicles and/or their composition and the propensity to form cholesterol crystals (nucleation time). Bile (common bile duct, gallbladder and T-tube) was obtained from patients with and without gallstones. Gel filtration chromatography resolved three peaks, a void volume vesicle, a smaller vesicle (identified by electron microscopy and of distinct composition compared to the larger void volume vesicle), and the mixed micelle. The void volume vesicle was present in 11 of 28 abnormal gallbladder biles, but in none of the 10 normal gallbladder biles. Despite this difference, no correlation between the nucleation time of whole bile with either the percentage of cholesterol carried by or cholesterol/phospholipid ratio of the void volume vesicle was found. Nucleation time was, however, found to correlate with the composition of the small-vesicular transport form. No significant difference in the composition or percentage of the small-vesicular form or the combined vesicular forms was found between normal and abnormal gallbladder biles, although the latter nucleated significantly more rapidly. Our results confirm the importance of vesicles in the nucleation process but suggest that other factors, not yet identified, appear to be responsible for the more rapid nucleation seen in abnormal gallbladder biles.