To generate broadly protective T cell responses more similar to those acquired after vaccination with radiation-attenuated Plasmodium falciparum sporozoites, we have constructed candidate subunit malaria vaccines expressing six preerythrocytic antigens linked together to produce a 3240-aa-long polyprotein (L3SEPTL). This polyprotein was expressed by a plasmid DNA vaccine vector (DNA) and by two attenuated poxvirus vectors, modified vaccinia virus Ankara (MVA) and fowlpox virus of the FP9 strain. MVAL3SEPTL boosted anti-thrombospondin-related adhesive protein (anti-TRAP) and anti-liver stage antigen 1 (anti-LSA1) CD8(+) T cell responses when primed by single antigen TRAP- or LSA1-expressing DNAs, respectively, but not by DNA-L3SEPTL. However, prime boost regimes involving two heterologous viral vectors expressing L3SEPTL induced a strong cellular response directed against an LSA1 peptide located in the C-terminal region of the polyprotein. Peptide-specific T cells secreted IFN-gamma and were cytotoxic. IFN-gamma-secreting T cells specific for each of the six antigens were induced after vaccination with L3SEPTL, supporting the use of polyprotein inserts to induce multispecific T cells against P. falciparum. The use of polyprotein constructs in nonreplicating poxviruses should broaden the target antigen range of vaccine-induced immunity and increase the number of potential epitopes available for immunogenetically diverse human populations.