Ecto-nucleotide pyrophosphatase/phosphodiesterase-I enzyme (E-NPP) consists of three closely related molecules: E-NPP1, E-NPP2 and E-NPP3. We investigated the expression and localization of E-NPP1 and -3 in human inflammatory and neoplastic bile duct diseases. Immunohistochemically E-NPP1 was located on the apical cytoplasmic side of cancer cells, whereas E-NPP3 was located in the apical plasma membrane. Western blot analysis revealed that the expression of E-NPP3, but not E-NPP1, was higher in tumor tissues than in surrounding tissues and the specific form of the E-NPP3 protein was readily detected in the sera of bile duct carcinoma (BDC) patients. Furthermore, it was confirmed that E-NPP3 was associated with migration ability by using of NIH3T3 cells that stably transfected with E-NPP3 cDNA. These results suggest that E-NPP3 is involved in the infiltration of neoplastic BDC and is possible to be a tumor marker.