Napsin A is a newly discovered functional aspartic proteinase that is expressed in normal lung parenchyma in type II pneumocytes and is thought to be associated with primary lung adenocarcinoma. Thyroid transcription factor-1 (TTF-1) is a widely used relatively restricted marker for lung adenocarcinoma. The present study aimed to compare the usefulness of napsin A with TTF-1 for the identification of primary lung adenocarcinoma. Immunohistochemical expression of napsin A and TTF-1 was analyzed in 351 lung cancer tissues, including 27 metastases. Napsin A was expressed in 180 of 212 (84.9%) primary lung adenocarcinomas, while no expression was noted in all 27 metastatic lung cancer specimens, including 19 metastatic adenocarcinomas. In contrast, TTF-1 expression was not only noted in 179 of 212 (84.4%) primary lung adenocarcinomas, but also in 12 of 18 (66.7%) small-cell carcinomas and some of the squamous carcinomas, as well as in one metastatic adenocarcinoma from the thyroid. The sensitivity and specificity of napsin A for primary lung adenocarcinoma (84.9 and 93.8%, respectively) were higher than the sensitivity and specificity of TTF-1 (84.4 and 83.9%, respectively). By combining napsin A and TTF-1, sensitivity increased to 91.0%. Furthermore, the sensitivity and specificity expression was associated with gender, smoking history, performance status, pathological type, primary tumor size and nodal metastasis. Therefore, napsin A is a useful novel marker in the differential diagnosis of primary lung adenocarcinoma.