Current theory suggests that the nucleation of cholesterol in human bile requires the aggregation and fusion of cholesterol-enriched phospholipid vesicles. This theory is based on observations which do not exclude the precipitation of cholesterol from mixed micelles. The present study examines the role of mixed micelles and vesicles in the formation of cholesterol monohydrate crystals in the prairie dog. The intermicellar bile salt concentration of prairie dog gallbladder bile was determined using equilibrium dialysis. Model bile equivalent to gallbladder bile from cholesterol-fed prairie dogs was used as dialysant yielding the intermicellar (dialysate) concentration of 9 mM. Cholesterol carriers in gallbladder bile from 11 cholesterol-fed animals were then separated by Sephacryl S200 gel filtration chromatography using eluant buffer containing the intermicellar bile salt concentration. Gel filtration chromatography of fresh bile demonstrated that 100% of cholesterol was carried in the mixed micellar fraction with no vesicles observed in any of the 11 animals. The gallbladder bile nucleation time was 2.0 +/- 0.3 days for the cholesterol-fed animals. Gel filtration chromatography immediately after nucleation again revealed a single mixed micellar peak. These data indicate that cholesterol is carried exclusively in and nucleates rapidly from mixed micelles in the cholesterol-fed prairie dog and that cholesterol-phospholipid vesicles are not required in this process.