Th1 effector T cells play a critical role in the host defense, cancer immunosurveillance, as well as a potential pathogenic role in autoimmune diseases. Interferon-γ (IFNγ) expression is critical for Th1 effector T cell priming and function. Although critical regulatory elements have been identified in the IFNγ promoter, little is known of the signal transduction mechanisms by which IFNγ is induced after activation via the T cell receptor (TCR) or cytokine receptors. We chose to examine the role of stress activated MAP kinases upon activation-induced transcription of the IFNγ gene. Expression plasmids encoding human dominant-negative (dn) p38 MAP kinase, JNK1 or JNK2 were cotransfected with a reporter plasmid containing the 538 bp IFNγ promoter linked to luciferase in transient transfection assays of human Jurkat T cells. Cells cotransfected with increasing concentrations of a dn p38 MAP kinase (with the total amount of expression vector held constant) showed a dose-responsive decrease, up to 70%, in IFNγ promoter-driven luciferase activity in cells stimulated with ionomycin and PMA (p < 0.007). Unstimulated cells had no luciferase activity. Dominant-negative JNK1 had no effect on the IFNγ promoter-driven luciferase activity in stimulated cells, serving as an internal control. Conversely, cotransfection of dn JNK2 enhanced luciferase activity by 75% in stimulated cells (p<0.02) compared to an equivalent amount of empty expression vector. In Jurkat T cells incubated with 0.1 μM to 10 μM of SB203580, a specific inhibitor of p38 MAP kinase, IFNγ promoter-driven luciferase activity was inhibited up to 70% in stimulated cells in a dose-responsive manner without a change of cell viability. Cotransfection with either upstream dual MAP kinase kinase molecules, MKK3 or MKK6, demonstrated that the constitutively active form of both of these molecules increased luciferase activity directed by the IFNγ promoter, up to 5 fold, while the dominant negative forms reduced expression by 50% in stimulated cells. These data implicate the stress-activated p38 MAP kinase to be critical for activation-induced transcription of the IFNγ gene.