DNA immunization is a very promising approach to the formulation of multivalent vaccines. However, little information is currently available on the immunogenicity of multi-plasmid formulations. To address this issue, we immunized mice with a combination of four plasmids encoding malarial antigens and we compared antibody responses with those obtained with single-plasmid injections. We found that when four plasmids encoding Plasmodium falciparum circumsporozoite protein, thrombospondin-related anonymous protein, major merozoite surface protein (MSP)1 and Pfs25 are co-injected into mice, Ab responses against each antigen are elicited at levels at least as high as the level obtained with single-plasmid injection. The quality of antibody production, as determined by isotype analysis, was similar when single- and multi-plasmid administrations were compared, indicating the priming of the same cytokine profile for CD4+ T helper cells. The sera from mice immunized with the four-plasmid formulation specifically recognized sporozoites, blood stage schizonts and gametes, indicating that DNA immunization induced antibody responses relevant to the native conformation. Finally and of particular interest, in the case of MSP1, the antibody response appears to be strongly potentiated by the presence of additional plasmids, indicating an adjuvant effect of DNA.