Because the epithelial cell adhesion molecule (Ep-CAM) is expressed in almost all carcinomas and human leucocyte antigen (HLA)-A*2402 is the most common allele in many ethnic groups, including Japanese, the identification of peptide sequences, which elicit HLA-A*2402-restricted Ep-CAM-specific cytotoxic T-lymphocyte (CTL) responses, would facilitate specific immunotherapy for various histological types of carcinomas. An epitope was identified through the following steps: (i) computer-based epitope prediction from the amino acid sequence of Ep-CAM, (ii) major histocompatibility complex (MHC) stabilization assay to determine the affinity of the predicted peptide with HLA-A*2402 molecules, (iii) stimulation of CD8(+) T cells with peptide-pulsed dendritic cells and (iv) testing the CTL specificity by means of enzyme-linked immunospot (ELISPOT) assays, CTL assays and MHC/peptide-tetramer staining. Peripheral CD8(+) T cells of four of five healthy donors after three rounds of stimulation with the peptide Ep-CAM(173-181) (RYQLDPKFI) secreted interferon-gamma in ELISPOT assays when exposed to the peptide. A CTL clone specific to the peptide efficiently lysed Ep-CAM-expressing cancer cell lines in an HLA-A*2402-restricted fashion. Endogenous processing and presentation of the peptide in a lung cancer cell line were confirmed by means of cold target inhibition assays. The CTL clone was also lytic to normal bronchial epithelial cells but to a lesser extent at low effector: target ratios. All these data suggest that the peptide-specific CTL responses may play some roles both in anti-cancer and autoimmune reactions. The peptide should prove useful to study anti-Ep-CAM CTL responses among population possessing HLA-A*2402.