The Staphylococcal enterotoxin superantigens stimulate vigorous responses in T cells bearing certain T cell antigen receptor (TCR) V beta regions. In addition to activation, these superantigens also impart negative signals to T cells resulting in a profound state of unresponsiveness or anergy. The Staphylococcus aureus enterotoxins (SE) B and C2 bind to a closely related site on major histocompatibility complex (MHC) human leukocyte antigen (HLA)-DR1 molecules. Only SEB, however, interacts with the TCR V beta 3 region of HA1.7, a human HLA-DR1 restricted T cell clone specific for influenza haemagglutinin. In competition experiments, we demonstrated that the induction of anergy in HA1.7 by SEB is unaffected by the presence of SEC2. These results suggest that SEB-induced anergy is MHC independent and involves a direct interaction between the TCR and SEB. To resolve definitively whether SEB binds directly to T cells in the absence of MHC class II molecules, the cDNAs encoding the HA1.7 TCR were transfected into an MHC class II-negative human T cell line. The addition of SEB to these transfectants resulted in the downregulation of cell surface TCR expression, an increase in the concentration of intracellular calcium ions, the production of lymphokines, and reduced responsiveness to a subsequent challenge with SEB. We conclude that SEB interacts directly with the TCR in the absence of cointeraction with MHC class II molecules, and that this interaction may induce anergy in HA1.7.