The prognosis of extrahepatic biliary tract cancer (EBT) patients is generally accepted to be poor. We immunohistochemically evaluated expression of p16, a cyclin-depend kinase inhibitor, in tumor specimens surgically removed from 99 EBT patients. We also examined whether there was any relationship between expression of p16 and biological malignancy of the tumor by comparing its clinicopathological factors. Consequently, we found that there were three types of p16 expression in the tumor cells; diffuse, heterogeneous and negative types, the percentages of which were 19, 41 and 39%, respectively. Heterogeneous and negative types, whose expression of p16 was considered to be down-regulated, showed scirrhous (p=0.022) and infiltrating growth (p=0.002). In addition, we found that the proportion of down-regulated expression of p16 was different, depending on the location of the tumor. We also observed that the down-regulated p16 expression was the highest in a proportion of patients with the extrahepatic bile duct carcinoma. In contrast, the proportion of down-regulated p16 expression was the least among the patients in the region of the ampulla of Vater with better prognosis, and we showed that the prognosis of patients with down-regulated expression of p16 was the poorest in terms of the cancer location where it is limited to the region of ampulla of Vater. These findings suggest that down-regulated p16 expression is evaluated as a factor of poorer prognosis and also that immunohistochemical pattern of p16 expression becomes a marker reflecting the biological malignancy of EBT patients.