We hypothesized that a high level of phospholipase A(2) (PLA(2)) in bile juice works as a growth factor of biliary epithelial cells and causes hyperplastic change of the bile duct in pancreaticobiliary maljunction. in order to examine this hypothesis, we established a primary culture system of biliary epithelial cells from the extra-hepatic bile duct of rats. We successfully excluded fibroblasts from the epithelial cells using D-valine-containing medium and the single-cell cloning method. The established cells exhibited the characteristic features of rat bile duct epithelial cells. They were stained with anti-cytokeratin antibody, a marker protein of epithelial cells, were positive with PAS stain, and possessed the architectures of microvilli, tonofilament fibers and desmosomes. The effect of PLA(2) on proliferation of these cells was then examined. We clearly demonstrated that PLA(2) at nano-molar concentration (50nM, 100nM), that is, equivalent to the level of PLA(2) in the bile juice of pancreaticobiliary maljunction cases, caused a significant enhancement of epithelial cell proliferation.