A defining characteristic of superantigens is their ability to stimulate T cells based predominantly on the type of variable segment of the T cell receptor (TCK) β chain (Vβ). The VB specificity of these toxins most likely results from direct contact between the toxin and the TCR, although the low affinity nature of this binding has prevented direct assessment of this interaction. To identify important functional sites on the toxin, we created chimeric enterotoxin genes between staphylococcal enterotoxins A and E (SEA and SEE) and tested the Vβ specificity of the chimeric toxins. This approach allowed us to identify three amino acid residues in the extreme COOH terminus of these toxins that are largely responsible for their ability to stimulate either human Vβ5- or Vβ8-bearing T cells, or mouse Vβ3 or Vβ11. We also found that residues in the NH2 terminus were required for wild-type levels of Vβ-specific T cell activation, suggesting that the NH2 and COOH ends of these superantigens may come together to form the full TCR Vβ contact site. SEA and SEE also differ with respect to their class II binding characteristics. Using the same chimeric molecules, we demonstrate that the first third of the molecule controls the class II binding phenotype. These data lead us to propose that for SEA and SEE, and perhaps for all bacterial-derived superantigens, the COOH and NH2 termini together form the contact sites for the TCR and therefore largely determine the Vβ specificity of the toxin, while the NH2 terminus alone binds major histocompatibility complex class II molecules. The predominant role of the COOH terminus of bacterial superantigens in determining Vβ specificity resembles current models being proposed for vitally encoded superantigens, suggesting that these molecules may demonstrate some structural relationship not seen at the amino acid level. © Rockefeller University Press.