The National Cooperative Gallstone Study was a double-masked trial conducted to determine the efficacy and safety of chenodeoxycholic acid (chenodiol) for dissolution of cholesterol gallstones. Patients with radiolucent gallstones were randomly allocated to either a high dose (750 mg/d, n = 305) or low dose (375 mg/d, n = 306) of chenodiol or placebo (n = 305) administered for 2 yr. Specimens of gallbladder bile were obtained for biliary lipid analysis on 50% of all white obtained for biliary lipid analysis on 50% of all white patients at base line and after 3-mo therapy, on 45% at 12 mo, and on 36% at 24 mo. Among these specimens, 20% were inadequate for analysis. For analysis of data, available values during therapy were averaged up to time of dissolution, study exit, or study termination. In the high-dose group, percent chenodiol (molar percent of all bile acids) increased markedly and remained high during the 2 yr of follow-up. Also, molar percent cholesterol decreased significantly and remained low during the 2 yr of follow-up. In the low-dose group, percent chenodiol increased and remained significantly increased. Percent cholesterol saturation decreased at 3 mo, but at 24 mo it was not different from that in the placebo group, suggesting a physiological adaptation to the low dose by 2 yr. 79% of patients on high dose had greater than 70% chenodiol. Among these, half showed unsaturated bile (less than 100% cholesterol saturation) while the remainder were supersaturated; in the former group with unsaturated bile, 23% had complete dissolution and 51% had partial (greater than 50% reduction in stone size) or complete dissolution. In contrast, those with over 70% chenodiol and supersaturated bile had only 5% complete dissolution. Thus, development of unsaturated bile was a major factor associated with gallstone dissolution. The data also indicate that values for percent cholesterol saturation were a better predictor of gallstone dissolution than molar percent chenodiol, although a high percent chenodiol usually was required to obtain unsaturation.