The Staphylococcal enterotoxin superantigens stimulate vigorous responses in T cells bearing certain T cell antigen receptor (TCR) V β 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)-DK1 molecules. Only SEB, however, interacts with the TCR Vβ3 region of HA1.7, a human HLA-DK1 restricted T cell done 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 TCK were transfected into an MHC class II-negative human T cell line. The addition of SEB to these transfectants resulted in the downregnlation of cell surface TCK expression, an increase in the concentration of intracellular calcium ions, the production of lympholcines, 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.