Calcium salts are often present in the center of all types of gallstones. Matrix proteins are known to be essential for biomineralization and may therefore also be important in the formation and growth of gallstones. Other researchers have described an anionic peptide fraction of a biliary lipoprotein complex in bile and a low—molecular weight acidic glycoprotein present in gallstones. Our goal was to determine whether such a protein was present in bile and whether this protein has any calcium–binding properties. We identified a pigment–associated, highly acidic protein that precipitates from bile on addition of CaCl2 0.5 mol/L. In addition, the protein is selectively concentrated in cholesterol and pigment stones. We have, therefore, confirmed the findings of these other researchers, and we have extended the study of this protein's interactions with calcium. Sodium dodecylsulfate—polyacrylamide gel electrophoresis demonstrates a single band (molecular weight ≤ 14 kD) that reacts positively with cationic stains. The protein was shown to inhibit the precipitation of CaCO3 from a supersaturated solution. The capacity to bind calcium was further confirmed by autoradiography with 45Ca++ and by a membrane adsorption—binding assay. Calcium–induced aggregation was demonstrated by equilibrium dialysis and by quasielastic light scattering studies. Protein measured by Lowry's assay method and amino acid analysis constitutes only 2% to 4% of the harvested material. We speculate that a substantial lipid component may also be present. The presence of this material in bile, its ability to bind pigment and calcium, its presumed lipid content, its self–aggregation in the presence of calcium and its selective incorporation into gallstones suggest that it may play a functional role in the pathogenesis of gallstones.