Sporozoite surface protein 2 has been identified as a target of malaria vaccines designed to produce protective CD8+ cytotoxic T lymphocytes (CTL) because mice immunized with mastocytoma cells expressing a fragment of Plasmodium yoelii sporozoite surface protein 2 (PySSP2) are protected against malaria by an immune response that requires CD8+ CTL. To define CTL epitopes in the Plasmodium falciparum sporozoite surface protein 2 (PfSSP2), spleen cells (SC) from mice immunized with irradiated sporozoites (irr spz) were stimulated with synthetic peptides, and these effectors were tested for cytolytic activity against peptide-pulsed, major histocompatibility complex (MHC)-matched targets. Two peptides containing CTL epitopes, A6 (Pf SSP2 3D7 214-233) and BH1 (Pf SSP2 3D7 3-11) were identified in bulk cultures of SC from immune C57BL/6 mice, and by production of CTL lines. Immunization with recombinant vaccinia expressing the full length PfSSP2 induced antigen specific, MHC-restricted, CD8+ T cell-dependent cytolytic activity against these two peptides. Finally, CTL were induced by immunization with a bacteria-derived recombinant fragment of PfSSP2 (rPfSSP2) mixed with a liposomal formulation containing a cationic lipid (Lipofectin Reagent, LPF). Induced CTL lysed target cells pulsed with peptide A6 or with LPF/rPfSSP2, but not targets pulsed with only rPfSSP2. These studies demonstrate that CTL specific to PfSSP2 are present in C57BL/6 mice and that immunization with purified rPfSSP2 delivered with LPF induces a cytotoxic T cell response.