We have recently shown that oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG motif) can induce B cells to proliferate, differentiate, and secrete cytokines. In this study we demonstrate that CpG motifs contained in ODN as short as 15 bases in length were quite effective at inducing NK cell lytic activity in vitro in both human and murine lymphocytes. Such ODN were also effective at inducing NK lytic activity, in vivo, in mice. Experiments designed to determine the cellular and cytokine requirements for NK cell induction revealed that B and T cells are not necessary, that the ODN do not augment the activity of highly purified NK cells, and that the ODN augment NK cell activity indirectly by inducing the secretion of IL-12, IFN-alpha beta, and TNF-alpha. Various ODN sequences were prepared to determine the optimal ODN length, motif, palindrome, backbone modification, and dose requirements. We found no requirement for a palindromic sequence but a definite requirement for an unmethylated CpG motif. While necessary, however, a CpG motif was not sufficient for NK cell induction. Instead, there appeared to be stringent requirements for the immediate flanking bases at the 5' and 3' ends as well as for flanking sequences outside the immediate 5' and 3' bases. In particular poly(G) ends seemed to exert a complex qualitative and quantitative effect which could be up- or down-modulating depending on whether the ODN backbone was phosphorothioate modified or not.