Identification of the cytotoxic T lymphocyte (CTL) epitopes of tumor antigens is important for effective immunotherapy. We report that a combination of epitope prediction, enzyme-linked immunosorbent assay (ELISA)-based epitope-HLA complex formation, and DNA immunization methods can improve the efficiency and accuracy of CTL epitope studies. In this study, two HLA-A11-restricted epitopes derived from human papillomavirus (HPV)18 E6 oncoprotein were identified. HLA-A11-transgenic mice immunized with these epitopes could specifically induce interferon-gamma (IFNgamma) production, cytotoxicity and peptide/HLA-A11 tetramer binding in CD8(+) T-cells. To study intracellular processing of CTL epitopes, we constructed a DNA plasmid containing an endoplasmic reticulum (ER) targeting sequence as well as the HPV18 E6 and E7 genes (pEK/HPV18E6E7). CTL responses against peptide-pulsed T2/A11 cells could be detected after immunizing HLA-A11-transgenic mice with pEK/HPV18E6E7. Furthermore, the identified peptides could stimulate T-cells to secrete IFNgamma from HPV18-infected patients. Our results demonstrate that the antigenic E6 peptides derived from HPV18 are potential candidates for the treatment of HPV 18-associated tumors in HLA-A11(+) populations.