CD10, a cell surface enzyme with neutral metalloendopeptidase activity, is a marker for intestinal epithelial brush border. It is also present in normal bile ducts and gallbladder epithelia but is absent in cholangiocarcinomas. However, the expression profile of CD10 in benign and malignant extrahepatic biliary lesions has not been studied. In this study, 69 biopsies, 9 resections, and 9 cell blocks prepared from fine-needle aspirations of the extrahepatic bile ducts from 86 patients were studied immunohistochemically for CD10 expression. The majority of cases contained normal biliary epithelium (NL, n=64), along with foci of benign or malignant lesions in various combinations. Benign lesions included reactive atypia (n=35), low-grade dysplasia of unknown significance (n=21), and bile duct adenoma (BDA, n=1). Malignant lesions included high-grade dysplasia (HGD, n=45) and invasive adenocarcinoma (IC, n=30). As expected, the NL showed strong continuous staining at the apical surface in all cases. Benign lesions were also CD10 positive in all but 3 cases; however, the staining pattern was discontinuous, with positive cells varying from 20% to 80%. None of the malignant lesions showed CD10 immunoreactivity, except for 2 HGD cases and 1 IC case, which exhibited focal staining. The Pearson χ2 and Fisher exact tests showed significant statistical difference in CD10 expression among the study groups (P<0.001). Our findings suggest that absence of CD10 expression in strips of atypical biliary epithelial cells may be a phenotype associated with malignant transformation and may serve as a useful marker to aid in the evaluation of bile duct biopsies, in which distinction between benign and malignant lesions on biopsies or cytology specimens can be extremely challenging because of limited sampling, crush artifact, and frequent inflammatory/reactive changes.