Toxic shock syndrome toxin 1 (TSST-1), a superantigen produced by Staphylococcus aureus, is a causative agent of toxic shock syndrome (TSS). This superantigen is a potent stimulator of T cells and macrophages/monocytes, resulting in the release of cytokines that are implicated in the pathogenesis of TSS. This study characterizes a mutant TSST-1, derived by site-directed mutagenesis, that has an alanine substitution at histidine 135 (mutant 135). This single-amino-acid change results in a mutant toxin that has lost mitogenic activity for T cells. In contrast to wild-type TSST-1, this mutant does not induce T cells to express interleukin-2, gamma interferon, or tumor necrosis factor beta (TNF-beta). The inability of mutant 135 to activate T cells is not due to a lack of binding to the class II major histocompatibility complex receptor. In addition, the mutant TSST-1 does not induce expression of TNF-alpha, which plays a role in the development of lethal shock. The lack of TNF-alpha induction by mutant 135 is likely due to its inability to activate T cells. These data suggest that the mutation at histidine 135 in TSST-1 affects toxin interactions with the T-cell receptor rather than the class II major histocompatibility complex receptor.