To examine the relation of cholesterol crystallization to the formation of gallstones, gallbladder bile was obtained by means of duodenal intubation in 54 patients (eight with asymptomatic gallstones) and from 17 patients undergoing cholecystectomy for cholelithiasis. Hepatic bile was obtained from nine patients with common-duct stones. Bile samples were examined for cholesterol monohydrate crystals and analyzed to determine the percentage of cholesterol saturation. Intubation in the eight patients with asymptomatic gallstones revealed a cholesterol saturation of 142 +/- 42 per cent (mean +/- S.D.) and crystals in five patients. Crystals were absent in the other 36 patients with supersaturated bile (cholesterol saturation, 166 +/- 44 per cent) and in the 10 patients with undersaturated bile (cholesterol saturation, 81 +/- 24 per cent). In the 26 patients with symptomatic cholelithiasis or common-duct stones, crystals were not seen in the bile of the seven patients with pigment stones but were present in the bile of all 19 patients with cholesterol stones. (In some cases crystals appeared only after 24 to 48 hours of incubation.) Cholesterol crystallization is probably a prerequisite for the formation of cholesterol gallstones; however, many subjects have no crystallization despite marked supersaturation.