Proteins associated with lipid vesicles or mixed micelles of human gallbladder bile were separated by Sepharose-2B gel filtration chromatography followed by protein concentration and delipidation. After two-dimensional polyacrylamide gel electrophoresis and silver staining up to 59 and 471 polypeptide spots were counted in vesicular and micellar fractions, respectively. As major components the plasma proteins transferrin, albumin, alpha-fibrinogen, beta-fibrinogen, gamma-immunoglobulin G, immunoglobulin light chains, alpha-1 antitrypsin and haptoglobin alpha-2 chain were identified in the lipid vesicles by comparison with human protein reference maps. However, most biliary proteins including the anionic polypeptide fraction are associated with mixed micelles. The pathophysiological significance of these proteins associated with lipids needs to be investigated further.