Immunization with DNA vaccines encoding relevant Ags can induce not only cell-mediated immune response but also humoral immune responses against pathogenic microorganisms in several animal models. Our previous results demonstrated that, when the C terminus (PyC2) of Plasmodium yoelii merozoite surface protein-1 (MSP-1), a leading vaccine candidate against erythrocytic stages of malaria, was expressed as a fusion protein (GST-PyC2) with glutathione S-transferase (GST), it elicited Ab-mediated protective immune responses in BALB/c mice. In our present study, we wished to examine the humoral responses to a DNA vaccine (V3) encoding GST-PyC2. The GST-PyC2 expressed in V3-transfected Cos 7 cells was recognized by a protective monoclonal Ab to PyC2 (mAb302), although the secreted product had undergone N-linked glycosylation. When BALB/c mice were immunized with V3 plasmid, anti-PyC2 Abs were successfully induced. These Abs immunoprecipitated native PyMSP-1 protein and competed with mAb302 for binding to its epitope at a level similar to those elicited by GST-PyC2 protein immunization. However, these Abs had significantly lower titers and avidities, and different isotype profiles and protective capacities against a lethal erythrocytic stage challenge, than those resulting from immunization with GST-PyC2 protein. Most surprising was the finding that, in contrast to protein immunization, there was no significant increase in the avidity of either GST-specific or PyC2-specific IgG Abs during the course of DNA immunization. This suggests that there may be little or no affinity maturation of specific Abs during DNA immunization in this system.