The MAGE-1 and MAGE-3 genes are members of the melanoma antigen-encoding gene family. These genes encode for HLA-restricted tumor-associated rejection antigens recognized by cytotoxic T lymphocytes. MAGE-1 and MAGE-3 gene expression has been identified in a number of human malignancies, and MAGE-1 and MAGE-3 antigenic peptides are potential targets for tumor-specific immunotherapy. The only normal tissues known to express these genes are testicular germ cells and placental tissue. The objective of this study was to examine MAGE-1 and MAGE-3 antigens immunohistochemically in testicular germ cell tumors, including seminoma, a germ cell tumor frequently associated with a lymphoid infiltrate. Forty-three germ cell tumors (24 seminomas, six embryonal carcinomas and 13 mixed germ cell tumors), and 10 Leydig cell tumors were selected for study, and standard immunohistochemical techniques were used on formalin-fixed paraffin-embedded tissues using mouse monoclonal antibodies to MAGE-1 (clone M454) and MAGE-3 (clone 57B) antigens. MAGE-1 antigen was identified in 16.6% of seminomas. No embryonal carcinomas, yolk sac tumors, or teratomas contained MAGE-1 protein. MAGE-3 antigen was identified in 41.8% of seminomas, and this protein was not identified in embryonal carcinomas, yolk sac tumors, or teratoma. Spermatogonia and primary spermatocytes contained MAGE-1 and MAGE-3 antigen, and more mature forms, including spermatids, were weakly positive to negative. Leydig cell tumors were negative for MAGE-1 and MAGE-3. In seminoma, the presence of MAGE-1 and MAGE-3 antigens did not correlate with tumor size, tumor stage, the presence of a lymphoid infiltrate, or patient outcome. The low frequency of MAGE-specific HLA alleles in the population, the loss of the HLA class I antigens in neoplastic germ cells, and the finding that the majority of seminomas and all non-seminomatous germ cell tumors lacked MAGE-1 and MAGE-3 antigenic peptides indicate that immunotherapy directed towards MAGE-1 and MAGE-3 antigen is not a likely treatment option for seminoma and nonseminomatous germ cell tumors. The significance of MAGE-1 and MAGE-3 proteins in normal spermatogonia and primary spermatocytes will require additional study.