The plasmid DNA vaccine not only provides expression of the antigen in vivo, but also activates cells of the innate immune system via unmethylated CpG-containing DNA sequences that are recognized by Toll like receptor 9 (TLR9). The requirement of such immunostimulatory activity for induction of CD8+ T-cell responses after DNA immunization is still controversial. In the present study we assessed induction of CD8+ T-cell responses against an immunodominant H-2D(b)-restricted epitope of human prostate-specific antigen in C57Bl/6 (wild-type), TLR9- and MyD88-deficient mice. A single DNA immunization resulted in efficient priming of CD8+ T responses in wild-type mice but not in TLR9- or MyD88-deficient mice. However, priming of CD8+ T cell responses was observed in TLR9-deficient but not in MyD88-deficient mice after multiple DNA immunizations. Moreover, induction of CD8+ T cell responses in TLR9-deficient mice was dependent on the presence of endotoxin contamination in plasmid DNA preparations. Collectively, these results demonstrate that TLR9-dependent immunostimulatory activity of plasmid DNA is essential for priming of CD8+ T-cell responses and that other bacterial compounds present in plasmid DNA preparations and acting via MyD88-dependent pathway could provide alternative signals necessary for priming of CD8+ T cells.