To elicit a cytotoxic T lymphocytes (CTL) response efficiently after DNA vaccination, we constructed several plasmid DNA (pDNA) vectors encoding the major histocompatibility complex (MHC) class I-restricted epitope peptide (SIINFEKL) of ovalbumin (OVA) or OVA protein with modified intracellular trafficking. An in vitro antigen presentation assay was carried out using DC2.4 cells, a dendritic cell line, to examine the potentials of the constructs following direct transfection. Among the vectors, pPep-ER, pDNA encoding antigen peptide combined with an endoplasmic reticulum (ER)-retention signal, exhibited a significant ability of antigen presentation compared with the counterpart without the signal. Based on the in vitro results, we carried out in vivo immunization experiments using pPep-ER via the intradermal or intramuscular route in combination with electroporation in mice. pPep-ER showed an efficient antigen-specific CTL induction and the effect was superior to that exhibited by the positive control, OVA in complete Freund's adjuvant (CFA). The levels of interferon gamma (IFN-γ) released from spleen cells were significantly increased by pPep-ER compared with pPep-free. Immunization with pPep-ER also exhibited a high inhibitory effect on the growth of E.G7 tumor. These results indicate that DNA vaccination with the pDNA vector expressing a MHC class I epitope peptide with controlled intracellular trafficking is a promising method of inducing an antigen-specific CTL response via direct presentation.