The study of cholesterol gallstone disease would be facilitated if the nucleation time of cholesterol crystals could be measured in duodenal bile and was correlated with nucleation occurring in vivo. Therefore, our aims were to determine (a) if nucleation time could be measured in duodenal bile, (b) the effect of bacteria, phospholipase, protease, and dilution on the measurement of nucleation time, and (c) the ability of nucleation time of duodenal bile to reflect changes occurring in vivo that promote the formation of gallstones and, therefore, the potential usefulness of nucleation time in predicting and studying the formation of gallstones. Gallbladder bile was obtained from 27 patients undergoing elective cholecystectomy and 19 patients undergoing diagnostic duodenal biliary drainage. Among the 14 bile samples collected by drainage that nucleated within 21 days, mean nucleation time was 6.3 +/- 2.8 days. The addition of inhibitors of phospholipase or protease prolonged nucleation time slightly. Bacteria were cultured from one bile sample at the time of collection and five samples at the time of nucleation. The addition of antibiotics had no effect on nucleation time. Dilution of bile collected at cholecystectomy to the concentration of duodenal bile prolonged nucleation time. In 4 of 5 obese patients receiving a very low calorie diet and predisposed to gallstones, the nucleation time in duodenal bile shortened, and the shortest nucleation times were associated with the formation of cholesterol crystals in vivo. Thus, measurement of nucleation time in duodenal bile may be useful in predicting and studying the formation of cholesterol gallstones.