We have recently demonstrated that linkage of DNA-encoding calreticulin to DNA-encoding human papillomavirus-16 E7 antigen strongly enhances the efficacy of DNA vaccines against E7-expressing tumors in animal models. In this study, as a prelude to clinical translation, we characterized the ability of DNA-encoding calreticulin linked to DNA-encoding E7 antigen to generate HLA-A2-restricted E7-specific CD8(+) T-cell responses in HLA-A2 (AAD) transgenic mice, as well as antitumor effects against an E7(+) HLA-A2(+) tumor cell line, TC-1/A2. Our results show that while vaccination with CRT/E7 DNA generates strong H-2D(b)-restricted E7 (amino acid (aa)49-57)-specific CD8(+) T-cell immune responses in both C57BL/6 and HLA-A2 (AAD) transgenic mice, no such responses were generated to HLA-A2-restricted epitopes in either type of mouse. In contrast, vaccination with DNA-encoding calreticulin linked to DNA encoding a mutant version of E7 with a deleted aa49-57 epitope leads to the generation of an HLA-A2-restricted E7 (aa11-20)-specific CTL response in HLA-A2 (AAD) transgenic mice. More importantly, vaccination with CRT/mtE7 (del aa49-57) DNA protects against a lethal challenge with TC-1/A2 tumor cells in HLA-A2 (AAD) transgenic mice. Furthermore, our in vitro studies demonstrate that the presence of the E7 (aa49-57) epitope does not suppress presentation of the HLA-A2-restricted E7 (aa11-20) epitope through MHC class I molecules. Thus, the predominant E7 aa49-57-specific CD8+ T-cell immune response in HLA-A2 transgenic mice vaccinated with CRT/E7 is likely due to preferred expansion of E7 aa49-57-specific CD8(+) T cells in vaccinated mice. These results highlight the importance of epitope immunodominance in the evaluation of immune responses in HLA-A2 (AAD) transgenic mice.