The presence of unmethylated CpG motifs in bacterial plasmids is thought to provide necessary immunoadjuvant signals to DNA vaccination. We took advantage of CpG-unresponsive toll-like receptor 9 (TLR9) knock-out mice to study whether this pathway was required to generate immune responses to DNA vaccination. We compared two vectors, one encoding the surface glycoprotein C of pseudorabies virus shown to protect target animals against challenge, and the other encoding the cytoplasmic enzyme beta-galactosidase. In the absence of TLR9, bone marrow-derived dendritic cells lost their ability to secrete IL-12 and type I IFN in response not only to CpG as expected but also to the plasmids used for vaccination. In contrast, DNA vaccination experiments showed that TLR9-deficient mice were able to mount Th1-biased antigen-specific antibody and IFN-gamma responses, albeit at lower levels than normal mice. Thus, TLR9 signaling is not needed for eliciting T- and B-cell responses to DNA encoded antigens. However, TLR9 signaling tended to enhance plasmid-adjuvant effects on antigen-specific immune responses.