Changes in gallbladder bile composition that occurred in patients who developed gallstones during weight reduction were evaluated. Bile was sampled directly from the gallbladder in 11 morbidly obese patients with no gallstones at the time of gastric bypass surgery and after gallstones had formed at cholecystectomy. Bile salt concentration ([BS]) increased significantly from a mean of 82.7-157.7 mmol/L (P less than 0.05). The concentration of cholesterol in gallbladder bile increased slightly and cholesterol saturation declined slightly with weight reduction and gallstone formation. Gallbladder mucin concentration increased 18-fold from a mean of 62 to 1110 micrograms/mL (P less than 0.001). Both free [Ca2+] and total calcium [Ca] increased 40% from mean values of 1.12 and 5.05 mmol/L at gastric bypass to 1.86 and 8.60 mmol/L after gallstone formation (P less than 0.05). The increase in [Ca2+] observed after gallstone formation was much greater than anticipated from changes in [BS] alone. This excess [Ca2+] in gallbladder bile increased curvilinearly with increasing mucin concentration. These results show that both gallbladder mucin and [Ca2+] increase with gallstone formation in humans and that mucin may modulate [Ca2+] in gallbladder bile.