Tumor antigens are the primary target of therapeutic cancer vaccines. We set out to define and compare the expression pattern of tumor antigen genes in esophagus carcinoma biopsies and in an allogeneic tumor lysate-based cancer vaccine, MelCancerVac. Cells used for vaccine production were treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) to determine whether this treatment could improve the profile of tumor antigen genes expressed in these cells. In addition, the presence of MAGE-A tumor antigen protein was evaluated in the purified tumor cell lysate used in the production of the vaccine. Quantitative PCR was used to assay 74 tumor antigen genes in patients with squamous cell carcinoma of the esophagus. 81% (13/16) of tumors expressed more than five cancer/testis (CT) antigens. A total of 96 genes were assayed in the tumor cell clone (DDM1.7) used to make tumor cell lysate for vaccine preparation. Gene expression in DDM1.7 cells was compared with three normal tissues; 16 tumor antigen genes were induced more than ten-fold relative to normal tissues. Treatment with 5-aza-CdR induced expression of an additional 15 tumor antigens to a total of 31. MAGE-A protein was detected in cell lysate by Western blot at an estimated concentration of 0.2 micrograms/ml or 0.01% of the total protein.