The liver is composed of a variety of cells that form a functional unit involved in uptake, synthesis, metabolism, and secretion. Until recently, most Studies examining liver function did not analyze the specific proteins expressed or functions performed by the multiple individual cell types that Constitute the hepatic mass. In the last decade, novel isolation methods have been developed that allow the purification of liver cell populations highly enriched in one type of liver Cell. Here, we present a detailed two- dimensional (2-D) protein map of rat bile duct epithelial cells (i.e., cholangiocytes) using a recently developed isolation procedure. In addition, we identify 27 major cholangiocyte proteins either by comparison to maps of known rat liver proteins (based on pI and M(r)) or by tryptic digestion and microsequencing. Finally, we compare the relative abundance of individual proteins present in cholangiocytes to whole liver as well as hepatocyte- specific proteins. Our results show that cholangiocytes express a unique array of individual proteins. The cholangiocyte 2-D protein pattern is markedly different from that of isolated rat hepatocytes or whole rat liver, with high levels of proteins previously known to be expressed by cholangiocytes (e.g., cytokeratins, actins) as well as protein not previously demonstrated to be expressed at high levels (e.g., annexin V, selenium binding protein). We conclude that this cholangiocyte-derived, 2-D protein map will be a crucial resource for studies directed at our understanding of cholangiocyte physiology and pathobiology.