Bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can trigger acute pathologic effects in humans. A hydrophobic loop on the surface of SEB and other bacterial superantigens, centered around a conserved leucine (L45) residue, is essential for binding to class II major histocompatibility complex molecules. Single residue changes of wild type SEB, designated Q43P, F44P, or L45R, resulted in nonlethal proteins at a dose equivalent to 30 murine LD50 of SEB. Relative to SEB, the mutant proteins did not elevate serum concentrations of proinflammatory cytokines in mice and caused minimal proliferation of human lymphocytes. Anti-SEB titers of mice immunized with Q43P, F44P, L45R, or SEB were similar and protected 77%-100% of animals against a lethal SEB challenge. Levels of toxin-specific IgG1, IgG2a, IgG2b, and IgG3 in mice immunized with SEB, Q43P, or F44P were equivalent, but animals immunized with L45R had significantly elevated levels of IgG2a and IgG2b. Vaccines against staphylococcal superantigens should focus on this critical leucine residue.