The staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1) induces massive cytokine production, which is believed to be the key factor in the pathogenesis of TSS. The temporal sequence and kinetics of both proinflammatory and anti-inflammatory cytokines induced by TSST-1 in human peripheral blood mononuclear cells were investigated. A panel of loss-of-function single-amino-acid-substitution mutants of TSST-1, previously demonstrated to be defective in either major histocompatibility complex (MHC) class II binding (G31R) or T-cell receptor (TCR) interaction (H135A, S14N), was studied in parallel to further elucidate the mechanisms of cytokine secretion. Wild-type recombinant (WT r) TSST-1 induced a biphasic pattern of cytokine secretion: an early phase with rapid release of proinflammatory cytokines (especially gamma interferon, interleukin-2 [IL-2], and tumor necrosis factor alpha [TNF-]) within 3 to 4 h poststimulation, and a later phase with more gradual production of both proinflammatory (IL-1, IL-12, and TNF-) and anti-inflammatory (IL-6, IL-10) cytokines within 16 to 72 h poststimulation. G31R, which is defective in MHC class II binding, induced a cytokine profile similar to that of WT rTSST-1, except that secretion of the early-phase proinflammatory cytokines was delayed and production of IL-1 and IL-12 was markedly reduced. In contrast, mutant toxins defective in TCR interaction either demonstrated complete absence of any cytokine secretion during the entire observation period (H135A) or resulted in complete abolishment of IL-2 and other early-phase proinflammatory cytokines, while secretion of IL-10 appeared unaffected (S14N). Neither WT rTSST-1 nor the mutant toxins induced IL-4 or transforming growth factor . Our data indicate that effective TCR interaction is critical for the induction of the early-phase proinflammatory cytokine response, thus underscoring the importance of T-cell signaling in TSS.