Thyroid transcription factor-1 (TTF-1) and Napsin-A (NapA) are frequently used to classify a tumor of unknown origin as lung or thyroid primary. Although recent studies have shown that nuclear TTF-1 positivity occasionally occurs in adenocarcinoma of nonpulmonary or thyroid origin dependent upon the antibody clone, TTF-1 has been reported as negative or infrequently positive in tumors of biliary origin. On the basis of an index case of cholangiocarcinoma expressing TTF-1, we were prompted to study TTF-1 and NapA positivity in cholangiocarcinoma. Archived paraffin-embedded tissue blocks from liver, gallbladder, and pancreato-biliary resections were chosen for cholangiocarcinoma (n=33) and non-neoplastic intrahepatic and extrahepatic biliary epithelium control tissue (n=26). Immunohistochemical analysis for TTF-1 and NapA was performed and graded for intensity and quantity. TTF-1 was negative in control biliary tissue but positive in 27.2% of cholangiocarcinomas. All TTF-1-positive cases (n=9) were extrahepatic (P=0.01), and most arose from the upper biliary tract (gallbladder and hepatic ducts). TTF-1 positivity was associated with age 60 years and above (P=0.01) but not with sex. Three TTF-1-positive cases were also NapA positive. NapA staining showed apical granular staining of the adjacent non-neoplastic epithelium in 6 cases (18.1%). In summary, 47.4% of extrahepatic cholangiocarcinoma expressed TTF-1, 33.3% of which coexpressed NapA. Cholangiocarcinoma should be considered in the differential when evaluating a TTF-1-positive tumor of unknown primary. As TTF-1 and NapA are not known for biliary system development or detected in non-neoplastic biliary epithelium, the significance of this "pulmonary" phenotype in a subset of extrahepatic cholangiocarcinoma is unknown and needs further investigation.