G astric adenocarcinomas carrying Epstein-Barr virus (EBV) are known to be accompanied by massive lymphocyte infiltration. To characterize the tumor-infiltrating lymphocytes (TILs), we isolated and cultured such cells from a surgically resected EBV-associated gastric carcinoma. They were found to be positive for CD3, CD8, T-cell receptor β chain, and cytotoxic molecules. The isolated TILs consisted of human leukocyte antigen (HLA) class I–restricted CD8⁺ cytotoxic T lymphocytes (CTLs), which killed autologous EBV-transformed cells (but not phytohemagglutinin blast cells) and recognized HLA-A24 as restriction molecules. However, the TILs did not recognize known EBV antigenic peptides presented by HLA-A24 molecules, nor HLA-A24⁺ fibroblasts infected with vaccinia recombinant virus expressing each of the EBV latent proteins. EBV⁺ gastric carcinomas do not express conventional target proteins of EBV-specific CTLs, and the data suggest that some cellular proteins may be involved in the strong T-cell response to EBV-associated gastric carcinoma. In addition, our data suggest that class I–restricted, antigen-specific CD8⁺ CTLs are specifically expanded within EBV⁺ gastric carcinoma tissue.