To study the microscopic and biochemical composition of biliary sludge, we subjected hepatic and gallbladder bile samples to microscopic examination, and analysis of lipids and mucus glycoprotein. These were compared with samples of hepatic and gallbladder bile obtained from normal persons and patients with gallstones. Biliary sludge was found to be sediment composed of cholesterol monohydrate crystals and bilirubin granules embedded in a matrix of mucus gel. There was a striking increase in the amount of mucus glycoprotein in the gallbladder bile of the sludge group compared with the gallstone and control group. The gallbladder biliary mucus content was also higher in the gallstone group compared with normal controls. These differences in mucus concentrations were not observed in hepatic bile samples from the three groups, suggesting that they had occurred within the gallbladder. In addition, gallbladder histology and epithelial mucin histochemical staining reaction were studied. Mucus hypersecretion and early glandular metaplasia in the epithelium were observed in the gallbladders harboring sludge, whereas those having stones had further changes in the mesenchymal tissues, with thickening and fibrosis. These results indicate that in patients with sludge the gallbladder is abnormal, showing mucus hypersecretion and glandular metaplasia. This leads to an increase in the mucus content of gallbladder bile, which in turn may result in nucleation of cholesterol crystals and may be regarded as the embryonic stage of gallstone disease.