We have recently characterized an MHC class II-deficient human cell line, SW480, that supports the proliferation of purified human T cells in the presence of the staphylococcal enterotoxin and superantigen SEC1, but not the closely related isotypes SEC2 or SEC3. We now investigate the structural basis of this dichotomy and explore possible mechanisms that may account for it. Differences in activity between SEC1 and SEC2 were not attributable to differences in biochemical modification, to differences in Vβ specificity, or to the potential to induce allergy. SEC2 inhibited SEC1-mediated T cell activation in the presence of SW480 cells, suggesting that SEC2 could compete with SEC1 for binding to the TCR but was unable to productively signal through the TCR. Utilizing a panel of hybrid enterotoxins we identified specific amino acids near the NH2-terminus of SEC1 that abrogated MHC class H-independent T cell activation, yet did not alter potency in the presence of class II+ APC. These residues mapped to the putative TCR binding domain of SEC1, and suggest that subtle differences in TCR binding affinity or the topology of the SEC1-TCR interaction can compensate for the lack of MHC class H and hence promote T cell proliferation.