NK and T cell-derived IFN-γ is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-γ, T-bet, IL-12Rβ2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-γ gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-γ mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-γ gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-γ gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-γ production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-γ gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15-and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-γ gene regulatory sites. IL-18, however, activated the binding of NF-κB to the IFN-γ promoter NF-κB site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.