We investigated whether bile concentration influenced cholesterol saturation index or nucleation time of cholesterol monohydrate crystals in a large number of gallbladder bile samples. Pigment stone patients never had cholesterol crystals in their fresh biles, and nucleation time was always longer than 20 days. Of the cholesterol stone patients 79% had cholesterol crystals in their fresh biles. Long nucleation times were generally found in cholesterol stone patients with dilute biles despite a high cholesterol saturation index. Nucleation time was usually short if bile was well concentrated despite a relatively low saturation index. Serial in vitro dilution of concentrated biles from cholesterol gallstone patients resulted in progressively prolonged nucleation times. Patients with solitary cholesterol stones had longer nucleation times than patients with multiple cholesterol stones. This study indicates that bile concentration is an important factor for nucleation time and cholesterol saturation index. Moreover, solitary and multiple cholesterol stones may have a different pathogenesis.