Tumor necrosis factor a (TNF-alpha) is a potent proinflammatory cytokine and plays a crucial role in early events of inflammation. TNF-alpha is primarily produced by monocytes and T lymphocytes. In particular, T-cell-derived TNF-alpha plays a critical role in autoimmune inflammation and superantigen-induced septic shock. However, little is known about the intracellular signaling pathways that regulate TNF expression in T cells. Here we show that extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38-mitogen-activated protein kinase (MAPK) pathways control the transcription and synthesis of TNF-alpha in A3.01 T cells that produce the cytokine upon T cell activation by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. Selective activation of each of the distinct MAPK pathways by expression of constitutively active kinases is sufficient for TNF-alpha promoter induction. Furthermore, blockage of all three pathways almost abolishes TPA/ionomycin-induced transcriptional activation of the TNF-alpha promoter. Selective inhibition of one or more MAPK pathways impairs TNF-alpha induction by TPA/ionomycin, indicating a cooperation between these signal transduction pathways. Our approach revealed that the MAPK kinase 6 (MKK6)/p38 pathway is involved in both transcriptional and posttranscriptional regulation of TNF expression. Moreover, analysis of the progressive 5' deletion mutants of the TNF-alpha promoter indicates that distinct promoter regions are targeted by either ERK-, JNK-, or p38-activating pathways. Thus, unlike what has been reported for other TNF-alpha-producing cells, all three MAPK pathways are critical and cooperate to regulate transcription of the TNF-alpha gene in T lymphocytes, suggesting a T-cell-specific regulation of the cytokine.