Abstract We analyzed the molecular mechanisms by which IL-12 and IL-18 induce transcriptional activity of the IFN-γ promoter in primary human CD4+ T cells. In transfection experiments, we found that IL-18 directly induces IFN-γ promoter activity, whereas significant activation with IL-12 required costimulation with αCD3/CD28. Furthermore, IL-12 caused in vivo protection of a STAT4 (−236) binding site, whereas stimulation with IL-18 or IL-12 plus αCD3/CD28 induced occupancy of a downstream AP-1 site. Mutation of this AP-1 site abrogated both IL-12- and IL-18-mediated promoter activation, whereas mutation of the STAT site inhibited IL-12-dependent activation. These data suggest that both AP-1 and STAT4 are required for IL-12-dependent IFN-γ promoter activity, whereas IL-18 causes direct activation via AP-1. This differential activation of the IFN-γ promoter gives further insights into molecular pathways governing Th1 T cell development and differentiation.