Toxic shock syndrome toxin 1 (TSST-1) contains a long central of helix that forms the base of two grooves on opposite sides of the molecule. Previous studies indicated that residues 132, 135, and 140 along the back of the central α helix are important in the biological activities. We made mutations of additional central α-helix residues exposed along this groove on the back of TSST-1. The proteins were purified, shown not to have gross alteration in structure, and tested for both superantigenicity and ability to elicit lethal TSS, using the miniosmotic pump model of TSS. Mutations of some residues along the central α helix resulted in decreased superantigenicity, likely because of alteration in T-cell receptor binding. Mutants H135A, Q136A, and E132K/ Q136K lost the ability to induce lethal TSS. The mutant Q136A was most interesting because it was superantigenic, yet nonlethal.