Changes in bile composition during cholesterol gallstone formation were investigated in hamsters. Gallstones were found in the gall-bladder and/or bile duct after feeding on a lithogenic diet for two weeks or more. Treatment with the lithogenic diet caused a marked increase in cholesterol secretion and a significant decrease in bile acid secretion, without affecting phospholipid secretion. The increase in cholesterol secretion was greater in gallstone-present than in gallstone-absent animals. Analysis of bile acids in the bile revealed that cholic acid, a major primary bile acid, was markedly decreased by the lithogenic diet. A decrease in glycine-conjugated bile acids and a relative increase in taurine-conjugated bile acids were also observed. The critical period for gallstone formation produced by the lithogenic diet was one to two weeks after the start of the lithogenic diet. From the above findings, the following mechanisms for cholesterol gallstone formation are suggested. The decrease in bile acid and a marked increase in cholesterol in the bile may result in the reduction of cholesterol solubility in micells consisting of bile acids, phospholipids and cholesterol. Subsequently, insoluble cholesterol forms gallstones. In addition, an alteration of glycine-conjugation capability may also play a part in gallstone formation.