Cytotoxic T lymphocytes (CTL) are key players in the neutralization of viruses and killing of tumor cells. However, for generating an optimal CTL response by vaccination, the antigen has to be delivered directly into the cytoplasm for presentation by the conventional MHC class I pathway. To mimic the presentation of multiple epitopes by a tumor or virus infected cell, we have designed a multiepitope peptide vaccine incorporating thee CTL epitopes in tandem with double arginine spacers to facilitate efficient cleavage of the individual epitopes. To deliver the multiepitope peptide vaccine into the cytoplasm of mature dendritic cells for presentation by the MHC class I pathway we made use of an amphipathic peptide carrier. Direct injection of a non-covalent complex of the multiepitope peptide vaccine and amphipathic peptide carrier in an aqueous formulation into HLA-A*0201 (HHD) transgenic mice enhanced the cytotoxic T-cell responses by two to sixfold compared with multiepitope peptide vaccination alone. This novel antigen delivery strategy may find general application in the development of more effective vaccines for the treatment of cancer and infectious disease.