We explored the influence of several compositional factors considered capable of influencing the nucleation time of model biles supersaturated in cholesterol. In addition to the classical techniques, e.g., electron microscopy and quasielastic light scattering, employed for size measurement and structural assessment, we employed a novel technique, i.e., video-enhanced microscopy, for particle evaluation in these polydisperse systems which often may simultaneously contain isolated small vesicles, their complex aggregates, and small cholesterol monohydrate crystals. The factors we studied included dilution, degree of cholesterol supersaturation, bile salt/lecithin molar ratio, and Ca2+ concentration. Dilution markedly raised the degree of cholesterol saturation, prolonged nucleation time for cholesterol monohydrate crystals, and favored formation of metastable small unilamellar vesicles. Increasing the degree of cholesterol supersaturation as an independent variable in more concentrated systems both shortened the nucleation time and favored spontaneous formation of a relatively small number of isolated vesicles. A decrease in bile salt/lecithin molar ratio within the physiologically relevant range was accompanied by a prolonged nucleation time and favored spontaneous vesicle formation. Large numbers of small unilamellar vesicles were observed even in concentrated model bile solutions (total lipids: 20 g/dl) when the bile salt/lecithin molar ratio was 1.9 or less. At physiological concentrations, Ca2+ promoted nucleation of cholesterol monohydrate crystals only in vesicle-containing solutions. Taken together, the following conclusions can be drawn. First, spontaneous vesicle formation in dilute systems prolongs solid cholesterol crystal nucleation. It can thus provide a supplementary non-micellar mode of cholesterol transport in micellar systems of supersaturated human bile. Second, dilution, degree of cholesterol supersaturation, and a decrease in bile salt/lecithin ratio prolong cholesterol crystal nucleation time and favor spontaneous vesicle formation. With increasing calcium concentrations, opposite effects are observed. Third, the presence of vesicles may help to account for the frequently observed and otherwise unexplained remarkable degree of metastable supersaturation and prolonged metastability (delayed nucleation time) for cholesterol in human bile.