We established gastric cancer-specific CD8+ T-cell (T(CD8+)) lines derived from different lymphocyte sources in the same patients by repeated stimulation with mitomycin-C-treated autologous tumor cells with low-dose interleukin-2, and we compared recognition patterns among the T(CD8+) derived from solid tumor, lymph node metastasis and ascites in the same patient (n = 3) to determine their similarities and differences for therapeutic purposes. We confirmed that gastric cancer-specific T(CD8+) lines can be isolated, in a MHC class I-restricted manner, from solid tumors, metastatic lymph nodes and malignant ascites. T(CD8+) lines derived from tumor-infiltrating lymphocytes (TIL) in solid tumor recognized autologous tumor cells derived from solid tumor, but not autologous tumor cells derived from ascites or metastatic lymph node, while T(CD8+) lines derived from tumor-associated lymphocytes (TAL) in malignant ascites recognized autologous tumor cells derived from ascites, but not tumor cells from solid tumor or metastatic lymph node. Furthermore, T(CD8+) lines derived from regional lymph node lymphocytes (RLNL) recognized autologous tumor cells derived from metastatic lymph nodes, but not tumor cells derived from ascites. No significant differences were seen in MHC class I expression among the tumors derived from solid tumor, lymph node metastasis or ascites in the same patient. This suggests that there are differences of recognition patterns among the TILs, TALs and RLNLs in the same patient and that it is important to consider the source of lymphocytes, e.g., a combination of TILs, TALs and RLNLs, for adoptive immunotherapy in gastric cancer patients.