XAGE-1 is a cancer/testis (CT) antigen and has been shown to be immunogenic in lung cancer patients. Among 4 alternative splicing variants, XAGE-1a, b, c and d, XAGE-1b mRNA was dominantly expressed in cancer. In this study, we generated a XAGE-1b mAb, USO9-13. The B cell epitope recognized by the USO9-13 mAb was in the C-terminal region of the XAGE-1b protein and is also recognized by sera from patients with lung adenocarcinoma. Using USO9-13 and an anti-Flag mAb, we examined the translation products of the 4 transcripts. The XAGE-1a and b transcripts translated to the XAGE-1b protein. The XAGE-1c transcript possibly translated to 9- and 17-aa polypeptides. The XAGE-1d transcript translated to a protein consisting of 69 amino acids. Immunofluorescence analysis using USO9-13 mAb showed that the XAGE-1b protein is located in the nuclei of cells. Immunohistochemically, nuclear staining was heterogeneously observed in 25/47 lung adenocarcinomas, 1/12 hepatocellular carcinomas and 1/11 gastric cancers, but not in adjacent normal tissues. These findings suggested that XAGE-1b is a promising target molecule for a cancer vaccine against lung cancer.