Precipitation of calcium salts from bile is important in pigment gallstone formation and may serve as a nidus for cholesterol precipitation. We compared gallbladder bile from patients with symptomatic gallstone disease (40 with cholesterol gallstones and 12 with pigment gallstones) with bile from 10 patients undergoing surgery for non-biliary tract disease. Bile from patients with gallstone disease was less concentrated, with decreased sodium, bile salt, and phospholipid concentrations, but elevated biliary calcium concentrations were not observed. The relationship between free ionized calcium and total calcium was similar in all groups, indicating no difference in calcium binding by gallstone-containing bile. We cannot exclude elevated biliary calcium level as a factor in gallstone pathogenesis, as it could be a transient event. The importance of calcium precipitation was supported by our finding that more than half of the samples were saturated or supersaturated with at least one calcium salt, calcium carbonate.