IL-12 is a central immunoregulatory cytokine that promotes cell-mediated immune responses and the differentiation of naive CD4+ cells into Th1 cells. We and others have demonstrated that the Stat4 is critical for IFN-gamma production by activated T cells and Th1 cells. However, several studies have suggested that other pathways may be involved in IL-12-stimulated IFN-gamma expression. In this report we demonstrate that IL-12 activates mitogen-activated protein kinase kinase 3/6 (MKK) and p38 mitogen-activated protein kinase (MAPK), but not p44/42 (ERK) or stress-activated protein kinase/c-Jun N-terminal kinase MAPK. The activation of p38 MAPK is required for normal induction of IFN-gamma mRNA and IFN-gamma secretion by IL-12 in activated T cells and Th1 cells. Importantly, IL-12-stimulated p38 MAPK effector functions occur through a Stat4-independent mechanism and correlate with increased serine phosphorylation of activating transcription factor-2. The requirement for p38 MAPK in IL-12 function suggests that this pathway may be an important in vivo target for the anti-inflammatory actions of p38 MAPK inhibitors.