The CD4-CD8+ CTL (KE-4 CTL) cell line against autologous tumor cells was established in a patient with esophageal cancer. This KE-4 CTL recognized a peptide antigen on esophageal and lung squamous cell carcinomas in an HLA A2601-restricted manner, as evaluated by cytotoxicity against a panel of tumor cells, transfection experiments with HLA A2601 cDNA, and reconstitution with eluted peptides. None of the normal cells tested was lysed by this CTL. These results suggest the existence of HLA A2601-restricted CTL precursors recognizing a peptide antigen on SCC in a patient with esophageal cancer.