Superantigens produced by Staphylococcus aureus and Streptococcus pyogenes are among the most lethal of toxins. Toxins in this large family trigger an excessive cellular immune response leading to toxic shock. Superantigens are secreted by the bacteria as diverse natural mixtures, a complexity that demands development of broad-spectrum countermeasures. We used a rational approach to design short peptides with homology to various domains in a typical superantigen (staphylococcal enterotoxin B) and screened each peptide for its ability to antagonize, in human peripheral blood mononuclear cells, superantigen-mediated induction of the genes encoding T helper 1 cytokines that mediate shock: interleukin-2, interferon-gamma and tumor necrosis factor. A dodecamer peptide proved a potent antagonist against widely different superantigens. This peptide protected mice from killing by superantigens and it was able to rescue mice undergoing toxic shock. The antagonist peptide shows homology to a beta-strand-hinge-alpha-helix domain that is structurally conserved among superantigens, yet currently of unknown function and remote from the binding sites for the known ligands essential for T cell activation, the major histocompatibility complex class II molecule and T cell receptor. The antagonist activity of this peptide thus identifies a novel domain in superantigens that is critical for their toxic action. The antagonist peptide provides a new tool for understanding the mechanism of excessive human immune response activation by superantigens that occurs during toxic shock and for identification of a novel target ligand that may interact with this superantigen domain.