A number of studies in animal models and humans have shown that both humoral andcell-mediated immune responses play an important role in the control of viral infection and tumor development. In most cases, vaccination with non-vectorized peptides or proteins induces low antibody responses and fails to elicit specific cytotoxic T lymphocytes (CTL). In order to make vaccination more efficient, we chemically coupled the non-toxic B subunit of Shiga toxin (STxB) to a full-size antigenic model protein, ovalbumin (OVA), yielding STxB-OVA. We found that STxB-OVA delivers OVA-derived peptides into both the MHC class I- and II-restricted presentation pathways in mouse dendritic cells. Accordingly, the study of STxB trafficking in these cells revealed that, after internalization, a fraction of STxB followed the retrograde transport pathway to the endoplasmic reticulum, while another fraction was targeted to late endosomes/lysosomes. Vaccination of mice with STxB-OVA primed a specific anti-OVA CTL response without the use of adjuvants. Splenocytes and, particularly, CD4(+) T cells from mice immunized with STxB-OVA also produced higher amounts of the T(h)1 cytokine IFN-gamma and IgG2a-type antibodies than mice immunized with non-vectorized ovalbumin. In conclusion, this study identifies a unique non-live vaccine delivery system for polyepitopic antigens that elicits antigen-specific CTL, a humoral immune response and a T(h)1-type polarization without the use of adjuvant.