Evidence from both animal and human viral diseases indicate that cytotoxic T lymphocytes (CTL) are crucial in antiviral defense. However, a major problem to generate cytotoxic immunity is that in vivo exogenous antigens are usually presented via MHC class II pathway and normally fail to induce CTL. The aim of this study is to describe a novel non-live prototype vaccine based on immunopotentiating reconstituted influenza virosomes (IRIV) as vehicles to deliver HLA-A*0201-restricted hepatitis C virus (HCV) peptides (core 35-44 and 131-140) into the cytoplasm of at least three different target cell types [including T2, a transporter associated with antigen processing (TAP)-deficient cell line] resulting in MHC class I peptide presentation and lysis by peptide-specific CTL lines. Comparison of kinetics and analysis of the influence of peptide-stripping and Brefeldin A (BFA) reveal that there exists an endogenous, TAP-independent and BFA-sensitive pathway for virosomally delivered peptides. Moreover, virosomes containing influenza matrix peptide 58-66 can efficiently re-stimulate in vivo primed CTL and, importantly, IRIV containing HCV core peptides can even prime CTL from peripheral blood mononuclear cells of HCV(-) healthy blood donors in vitro. The fact that in vitro primed CTL are also able to specifically lyse target cells infected with recombinant vaccinia virus encoding the HCV core protein is of great importance for future studies based on in vivo mouse models. One of the most evident advantages of the virosomes in vivo will be their capability to protect the incorporated peptide from a large variety of degrading proteases.