BACKGROUND Previous studies have shown that human bile contains one or more factors that inhibit the precipitation of calcium carbonate from supersaturated solutions of calcium chloride and sodium bicarbonate. Lower concentrations of this factor have been observed in the gallbladder bile of patients with calcified gallstones. We hypothesized that gallbladder bile contains factors that inhibit calcium carbonate and these factors are present in varying concentrations in normal persons and in patients with cholesterol gallstones with and without calcium carbonate. METHODS Gallbladder bile of patients without gallstones (n = 8) and of patients with cholesterol gallstones containing either calcium carbonate (n = 8) or other calcium salts (n = 8) was assayed for calcium carbonate inhibition. Individual components of bile (bilirubin, phospholipid, bile salts, and albumin) were tested in different concentrations in the same assay system. In addition, samples of model bile were tested. RESULTS An inhibitory factor for calcium carbonate precipitation was present within all human gallbladder bile, irrespective of the absence, presence, or type of gallstones. The addition of a bilirubin-albumin solution to a supersaturated solution of calcium chloride and sodium bicarbonate entirely blocked precipitation of calcium carbonate from solution. In addition, serial dilutions of bilirubin exhibited a linear response between bilirubin concentration and inhibitory effect. Model bile and phospholipid dissolved in sodium taurocholate also exhibited a modest inhibitory effect on calcium carbonate precipitation. CONCLUSIONS We conclude that bilirubin, cholesterol, and phospholipids incrementally interfere with calcium carbonate precipitation in supersaturated solutions through the preferential formation of a soluble calcium complex.