Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG ODN) mimic the immunostimulatory activity of microbial DNA via Toll-like receptor (TLR)9. Previous studies indicated that human NK cells express functional TLR3 and TLR9, since their cytokine release and cytolytic function could be incremented by poly(I:C) or ODN A/B, respectively. We have now evaluated the capability of a novel class of CpG ODN, termed ODN C, to modulate the function of human NK cells in the presence of exogenous cytokines. We show that NK cells isolated from peripheral blood and cultured with ODN C, in the presence of either IL-12 or IL-8, express higher levels of CD69 as compared to those stimulated with either ODN A or ODN B. Moreover, NK cells cultured with ODN C displayed higher cytolytic activity against tumor cell lines. These effects were not confined to freshly isolated peripheral blood NK cells since polyclonal NK cell populations that had been cultured in the presence of exogenous IL-2 for several weeks also displayed higher cytolytic activity and cytokine release after culture in the presence of ODN C. Remarkably, NK cells displaying poor responses to ODN A/B were efficiently stimulated by ODN C.