NK cells play an important role in innate immune resistance, particularly through synthesis of the pro-inflammatory cytokine IFN-γ. This study compares the abilities of the cytokine IL-18 and the costimulatory cell surface molecule CD28 to enhance IL-12-driven IFN-γ production by NK cells. Studies with other cytokines (IL-1β, IL-6, TNF-α, IL-15) showed that IL-18 or anti-CD28 treatments were the most efficient inducers of IFN-γ when combined with IL-12. The ability of IL-18 to enhance IFN-γ was shown to be dependent on the presence of IL-12. Similarly, although anti-CD28 stimulation alone could enhance IFN-γ synthesis, this effect was significantly increased in the presence of IL-12. Although neither method of costimulation required de novo protein synthesis for their effects on IFN-γ mRNA expression, these molecules used distinct mechanisms. Specifically, nuclear run-on analysis revealed that IL-18 in combination with IL-12 enhanced the rate of transcription of the IFN-γ gene. Conversely, treatment with anti-CD28 plus IL-12 did not significantly up-regulate the rate of transcription of the IFN-γ gene, but stabilized IFN-γ mRNA expression within NK cells. These findings illustrate costimulatory pathways that result in potent IFN-γ responses by NK cells and show that although IL-18 and anti-CD28 can enhance the synthesis of IL-12-driven IFN-γ, they employ molecular mechanisms that are distinct from one another.