Two very similar small anionic, amphipathic proteins, a phospholipid-binding apoprotein (anionic polypeptide fraction [APF]) and a calcium-binding polypeptide (CBP), are found abundantly in bile and all types of gallstones. The often disparate properties among various preparations of APF/CBP could reflect different sources and separation procedures, leading to partly degraded and/or denatured protein and varied association of bile salts, lipids, bile pigments, and detergents. The present study presents new methods for isolation and purification of APF/CBP, and characterizes the preparations thus obtained. It was found that isolation by selective precipitation of proteins from fresh T-tube bile by added calcium chloride, followed by demineralization with ethylenediaminetetraacetic acid (EDTA), removal of salts, lipids, and some pigment by Sephadex LH-20, and serial ultrafiltration yields the purest preparations. Though free of lipids, bile salts, detergents, and most pigments, these new preparations all show the same 7-kd and 12-kd bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the same major peaks on hydrophobic high-performance liquid chromatography (HPLC), and retain the self-associative, lipid- and calcium-binding functions, typical of older preparations obtained by potentially denaturative procedures. The varied properties among APF/CBP preparations are thus apparently related mainly to their content of different proportions of two major components, lipid-binding APF and calcium-binding CBP. Immunologic cross-reactions indicate common epitopes, and amino acid analyses are also similar, suggesting that APF and CBP may have the same polypeptide backbone, but differ because of posttranslational modification(s). Sufficiently pure APF and CBP have now been obtained to permit possible structural identification by sequencing and molecular biological techniques, though such attempts have thus far been unsuccessful.