Rapid nucleation of cholesterol crystals has previously been shown to provide a sharp discrimination between abnormal (cholesterol gallstone-associated) and normal human gallbladder bile. In the present study, we sought to further clarify the crystal nucleation process by time-lapse microscopy using a novel high-resolution video-enhanced microscopy technique. Using a previously described method for removal of particles from abnormal biles, we found a strikingly rapid rate of de novo formation of unilamellar vesicles, soon followed by massive vesicular aggregation, culminating in crystal formation. In normal biles, by contrast, this rapid aggregation process was not observed and the isolated unilamellar vesicles showed prolonged stability. Morphometric analysis of interval particle counts showed statistically significant differences. The process of cholesterol monohydrate crystal nucleation in supersaturated human bile is characterized by a sequential combination of vesicle formation, vesicle aggregation, and subsequent crystal formation. The primary distinction between abnormal and normal biles resides only in the consistent rapidity of onset and completion of these events in the abnormal biles.