Toxic shock syndrome toxin-1 (TSST-1) is a member of the staphylococcal enterotoxin superantigen family. In order to determine the regions on the TSST-1 molecule involved in binding to class II MHC, seven overlapping peptides of the entire TSST-1 molecule were synthesized and tested for their ability to compete with 125I-TSST-1 for binding to class II MHC on murine A20 cells and HLA on Raji cells. Peptides corresponding to N-terminal amino acid residues 39 through 68 and C-terminal residues 155 through 194 competed with 125I-TSST-1 for binding to class II MHC. Also, binding studies with class II MHC β-chain peptides indicate that regions encompassed by I- A(β)b(30-60) and I-A(β)b(60-90) are binding regions for TSST-1. Thus, we have identified binding domains on the TSST-1 molecule for class II MHC molecule receptors on antigen presenting cells. © 1993 Academic Press, Inc.