[show abstract][hide abstract] ABSTRACT: Plasmid DNA continues to attract interest as a potential vaccine-delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll-like receptor 9-deficient (TLR9−/−) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen-specific antibody and at stimulating antigen-specific interferon-γ (IFN-γ)-secreting cells in TLR9−/− mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.