Superantigens, such as staphylococcal enterotoxins (SE), cause food poisoning and shock, which is mediated at least in part by TNF. We have examined the mechanism of superantigen-induced activation of the TNF gene promoter in primary human peripheral blood T lymphocytes and in the leukemic T cell line Jurkat. Like stimulation of the T cell receptor complex through CD3 ligation, SEB induces binding of nuclear proteins to Egr- and Jun/ATF-related consensus sequences present between nucleotides -170 and -100 of the TNF promoter 5' flanking region. By cotransfection of Jurkat T cells with constructs containing TNF promoter deletion mutants linked to a CAT reporter gene, it is shown that superantigens can activate transcription from these two adjacent ETs and Jun/ATF binding elements. Superantigen-induced binding of Egr-1 and Jun/ATF is markedly reduced by okadaic acid, suggesting that phosphatases are involved in the signaling of SE. When compared to CD3 ligation, superantigens activate the TNF promoter more potently, which is likely due to costimulatory signals provided by superantigen presenting cells.