Several studies have suggested that there is a direct interaction between the T cell receptor (TCR) and the major histocompatibility complex (MHC) molecule during T cell recognition of superantigen. To further investigate this possibility, we have analyzed T cell recognition of a bacterial superantigen, Staphylococcal enterotoxin B (SEB), presented by a series of mutant murine I-Ek molecules in which residues of either the alpha or beta chain predicted to interact with the TCR have been substituted. Individual T cell hybridomas gave distinct patterns of responsiveness to SEB presented by the I-E beta k mutants that could not be attributed to differences in the binding of SEB to the mutants. This effect appeared to be dependent on the TCR-alpha chain because some of these hybridomas expressed identical TCR transgenic beta chains. In contrast, none of the hybridomas gave distinct patterns of responsiveness to SEB presented by the I-E alpha k mutants. Taken together, these observations support the idea that there is a functional interaction between the alpha chain of the TCR and the beta chain of the MHC class II molecule. The data also support the idea that this interaction might enhance superantigen recognition in some cases.