Vaccination with antigenic peptide-pulsed antigen-presenting cells (APC) represents an attractive approach for therapy for cancer and diseases caused by intracellular infections. It has been suggested that sufficient stable MHC/peptide complexes on the surface of APC might play an important role in the generation of antitumor and antiviral immunity in vivo. In this study, we observed that exogenous peptides that were artificially fused with an endoplasmic reticulum (ER) retrieval signal, a C-terminal Lys-Asp-Glu-Leu sequence, could be efficiently presented by intracellular MHC class I molecules in a TAP- and proteasome-independent, but brefeldin A-sensitive manner. The APC retained the capacity to display surface MHC/peptide complexes for a prolonged period. In addition, our results show that vaccination with DC bearing our fusion peptides induced greatly enhanced specific CTL response, and resulted in significant inhibition of tumor growth. Thus, the ER retrieval signal modification can be regarded as a novel method for targeting exogenous peptides into the intracellular MHC class I presentation pathway, and may improve the clinical utility of vaccines based on synthetic peptide pulsed DC.