IL-12 and IL-18 synergistically enhance IFN-gamma mRNA transcription by activating STAT4 and AP-1, respectively. However, it is still unknown how STAT4/AP-1 elicit IFN-gamma promoter activation. Using an IL-12/IL-18-responsive T cell clone, we investigated the mechanisms underlying synergistic enhancement of IFN-gamma mRNA expression induced by these two cytokines. Synergy was observed in a reporter gene assay using an IFN-gamma promoter fragment that binds AP-1, but not STAT4. An increase in c-Jun, a component of AP-1, in the nuclear compartment was elicited by stimulation with either IL-12 or IL-18, but accumulation of serine-phosphorylated c-Jun was induced only by IL-18 capable of activating c-Jun N-terminal kinase. The binding of AP-1 to the relevant promoter sequence depended on the presence of STAT4. STAT4 bound with c-Jun, and a phosphorylated c-Jun-STAT4 complex most efficiently interacted with the AP-1-relevant promoter sequence. Enhanced cobinding of STAT4 and c-Jun to the AP-1 sequence was also observed when activated lymph node T cells were exposed to IL-12 plus IL-18. These results show that STAT4 up-regulates AP-1-mediated IFN-gamma promoter activation without directly binding to the promoter sequence, providing a mechanistic explanation for IL-12/IL-18-induced synergistic enhancement of IFN-gamma gene expression.