A study was performed to determine whether the rapid nucleation time of gallbladder bile obtained from patients with cholesterol gallstones was due to the addition of a nucleating agent or the removal of an antinucleating agent by the gallbladder. Isotropic phases of gallbladder bile from normal controls (control bile) and from patients with gallstones (abnormal bile) were mixed 50:50 (vol/vol) and the nucleation times of the mixtures and parent biles were determined. The mixtures had rapid nucleation times, similar to those of the gallbladder bile from gallstone patients, indicating that a nucleating factor was present in the abnormal bile. Experiments were then performed using mixtures in which the proportion of abnormal bile was reduced. These studies showed that the nucleating agent was potent. The results were not due to changes in cholesterol saturation or total lipid concentration. The conclusions reached in the first study were supported in a second set of similar experiments in which hepatic bile from gallstone patients was mixed with their own gallbladder bile. It was also found that filtration of abnormal bile through an XM-300 Amicon filter did not eliminate its nucleating potency, indicating that the results could not be explained by the presence of residual microcrystals in the abnormal bile.