When modern methods of estimating cholesterol saturation in bile were first applied to human bile samples, it seemed that a clear distinction could be made between normal and abnormal biles on the basis of cholesterol saturation. Supersaturation of bile with cholesterol appeared to be the only critical defect required for stone formation. A variety of evidence has now accumulated which indicates that a nucleation defect is also important. Chief among these observations is the fact that gallbladder bile from stone patients nucleates cholesterol crystals more rapidly than equally saturated gallbladder bile from controls. To determine whether the gallbladder contributes to the nucleation defect, hepatic and gallbladder bile nucleation times have been compared. Although the former is more supersaturated, the latter nucleates faster suggesting that the gallbladder is important. In another study, the nucleation times of crystal-free abnormal bile, normal bile and a mixture of the two were obtained. The mixture nucleated as rapidly as the abnormal bile even when the abnormal bile contributed only 5% of the volume of the mixture. This indicated that a nucleating factor is added to abnormal bile in the gallbladder.