Nucleation of cholesterol crystals is thought to occur from cholesterol-phospholipid vesicles. We tested the hypothesis that bile acids are necessary for nucleation of cholesterol crystals. Model bile vesicles were prepared by KBr density ultracentrifugation of supersaturated model bile and mixed with one of the following bile acids: ursodeoxycholate, taurocholate, cholate, chenodeoxycholate or deoxycholate in final concentrations of 3, 30 and 100 mM. Vesicles were also mixed with various combinations of ursodeoxycholate and deoxycholate. Nucleation was assessed semi-quantitatively with polarizing microscopy. After 5 days, samples were again subjected to ultracentrifugation. Addition of 3 and 30 mM taurocholate, cholate, chenodeoxycholate and deoxycholate to vesicles induced nucleation. The extent of nucleation increased significantly with increasing bile acid hydrophobicity: deoxycholate > chenodeoxycholate > cholate > taurocholate (p < 0.05). At 100 mM bile acid this order was reversed (p < 0.05) because most of the cholesterol was solubilized in micelles as shown by ultracentrifugation after 5 days. Percentages of vesicular cholesterol decreased with increasing hydrophobicity: deoxycholate < chenodeoxycholate < cholate < taurocholate (p < 0.05). Ursodeoxycholate did not induce nucleation. At least seven cholesterol crystal shapes could be distinguished and all crystal types could be found after addition of various combinations of ursodeoxycholate+deoxycholate. We conclude that in this model: (a) bile acid species play an important role in the precipitation of cholesterol crystals from model bile vesicles; (b) the more hydrophobic bile acids induce more cholesterol crystal precipitation; and (c) the hydrophobicity of bile acids influences cholesterol crystal morphology.