Previous studies have shown that the action of bacterial or synthetic oligodeoxynucleotide (oligo-DNA) on mouse NK cells to produce IFN-γ is mediated mostly by monocytes/macrophages activated by olig-DNA. However, its action on human IFN-γ-producing cells has not been well investigated. In the present study, we examined the effect of oligo-DNAs on highly purified human NK and T cells. Bacillus Calmette-Guérin-derived or synthetic oligo-DNAs induced NK cells to produce IFN-γ with an increased CD69 expression, and the autocrine IFN-γ enhanced their cytotoxicity. The response of NK cells to oligo-DNAs was enhanced when the cells were activated with IL-2, IL-12, or anti-CD16 Ab. T cells did not produce IFN-γ in response to oligo-DNAs but did respond independently of IL-2 when they were stimulated with anti-CD3 Ab. In the action of oligo-DNAs, the palindrome sequence containing unmethylated 5′-CpG-3′ motif (s) appeared to play an important role in the IFN-γ-producing ability of NK cells. The changes of base composition inside or outside the palindrome sequence altered its activity: The homooligo-G-flanked GACGATCGTC was the most potent IFN-γ inducer for NK cells. The CG palindrome was also important for activated NK and T cells in their IFN-γ production, although certain nonpalindromes acted on them. Among the sequences tested, cell activation-or cell lineage-specific sequences were likely; ie, palindrome ACCGGT and nonpalindrome AACGAT were favored by activated NK cells but not by unactivated NK cells or activated T cells. These results indicate that oligo-DNAs containing CG palindrome act directly on human NK cells and activated T cells to induce IFN-γ production.