Gastric cancer has the highest mortality rate and the second-highest morbidity rate of all malignant tumors in China. Since cancer/testis (CT) antigens are expressed in various types of human tumors but generally not in normal tissue except for testis, they are promising antigens for cancer immunotherapy. NY-ESO-1, in particular, is the most immunogenic of the CT antigens. To study the feasibility of developing a CT antigen vaccine for gastric cancer, 101 gastric cancer samples were analyzed for the presence of NY-ESO-1 mRNA and that of 10 other CT antigen genes. Twelve out of 101 samples (11.9%) were found to be NY-ESO-1 mRNA-positive, 11 of them from advanced stage patients. In 7 of the 12 NY-ESO-1 mRNA-positive samples, the NY-ESO-1 protein was also detected by immunohistochemistry. An autologous humoral immune response to NY-ESO-1 was detected in 6 of 12 advanced stage NY-ESO-1 mRNA-positive patients, indicating that NY-ESO-1 is immunogenic in advanced stage gastric cancer. The serum from a patient with an NY-ESO-1 negative but LAGE-1 positive tumor was also found to be NY-ESO-1 antibody positive, possibly due to cross-reactivity between NY-ESO-1 and LAGE-1. All NY-ESO-1 mRNA-positive gastric cancer samples also expressed one to seven additional CT genes, revealing a tendency toward a clustered expression pattern, regardless of disease stage. About 74% of the samples expressed at least one CT antigen, most frequently MAGE-3 (41.6%). NY-ESO-1 and MAGE-3 are thus potential targets for a multivalent CT antigen vaccine.