Nocturnal hepatic bile is supersaturated with cholesterol, and increased storage of this "lithogenic" bile in gallstone patients might explain why they more frequently have supersaturated fasting state gallbladder bile than patients without gallstone disease. To test this possibility, we measured the fraction of nocturnal hepatic bile stored in the gallbladder in 5 patients whose gallstones had been previously dissolved by chenodeoxycholic acid, in 5 control subjects without gallstone disease matched for age, sex, and weight, and in 5 young healthy males. We developed a new method based on the use of indocyanine green as a bile marker. The hourly rate of input into the duodenum of indocyanine green was measured during its constant, prolonged intravenous infusion. When gallbladder storage occurred, indocyanine green input was less than the infusion rate; when gallbladder emptying occurred, input exceeded the infusion rate. The method was validated by showing that duodenal output of indocyanine green was identical to intravenous input of indocyanine green in 2 cholecystectomized patients. When the method was applied, emptying of the gallbladder with meals was documented, and calculations showed that more than half of the bile secreted at night bypassed the gallbladder in both gallstone patients and control subjects. No difference in "cholecystodynamics" was observed between gallstone patients, matched control subjects, and young healthy men. Our results validate a method for quantitating gallbladder storage and emptying in men, but fail to support the speculation that an important pathogenetic step in gallstone formation is increased storage of supersaturated nocturnal bile in the gallbladder.