Bacterial DNA differs from mammalian DNA by the presence of unmethylated cytosine-phosphate-guanosine (CpG) motifs. The immunostimulatory properties of a DNA vaccine have been suspected to be associated with these motifs. The aim of this study was to assess the inactivation of the immunostimulatory potential of a plasmid after methylation of its CpG motifs. We constructed two identical non-coding plasmids, and one of these was de novo methylated on its CG sequences. A single administration of recombinant antigen with methylated or unmethylated CpG-containing plasmid was performed in mice. As expected, only unmethylated CpG-containing plasmid enhanced the specific immune response. However, a study of in vivo activation of Langerhans' cells and analysis of mRNA synthesis indicated that both the plasmids promoted cell emigration and cytokine induction. These data highlight that a methylated CpG-containing plasmid is not inert and carries immunomodulatory properties. The results further emphasize the necessity to definitively identify the mode of action of plasmids used for DNA vaccination.