DNA vaccines, in general, have been found to be poorly immunogenic in nonhuman primates and humans as compared with mice. As the immunogenicity of DNA plasmids relies, to a large extent, on the presence of CpG motifs as built in adjuvants, we addressed the issue of poor immunogenicity by inserting recently identified CpG oligonucleotides (ODN) optimal for human (K-type or D-type CpG ODN) into the backbone of plasmid VR1020. We found that plasmid DNA containing K-type CpG motifs or D-type CpG motifs significantly enhanced the up-regulation of surface molecules and production of interleukin-6 from human peripheral blood mononuclear cells (PBMC) and stimulated monocytes to develop into functionally mature dendritic cells (DC) compared with unmodified plasmid. Monocyte maturation into DC was through plasmacytoid DC present in the culture. It is interesting that the K-type CpG motif-modified plasmid stimulated significant levels of interferon (IFN)-gamma and IFN-alpha from human PBMC. Immunization of mice with D-type CpG motif-modified plasmid, encoding Plasmodium falciparum surface protein 25, yielded enhanced antigen-specific antibodies. Taken together, these results suggest that insertion of immunomodulatory human CpG motifs into plasmid DNA can improve immunogenicity of DNA vaccines.