Previous studies identified three COOH-terminal residues in staphylococcal enterotoxin E (SEE; Asp200, Pro206, and Asp207) that in part mediate TCR Vβ recognition. We have identified an additional three residues near the NH2 terminus of SEE (Arg20, Asn21, and Ser24) that are needed in conjunction with these COOH-terminal residues to fully restore native levels of Vβ-specific T cell proliferation. A staphylococcal enterotoxin A (SEA)- SEE hybrid molecule containing the NH2-terminal Vβ determinants of SEE alone exhibited Vβ specificities of both SEA and SEE, indicating that these residues of SEE independently contribute to Vβ recognition and do not obscure the native Vβ determinants of SEA. These findings suggest that the ability of SEE to activate certain Vβ-specific T cell subsets may result from multiple interactions with a single TCR β-chain or perhaps by cross- linking two TCR. High affinity binding to HLA-DR1, a property of native SEA, was not altered in the SEA-SEE hybrid enterotoxins containing amino acid substitutions in regions 20 to 24 and 200 to 207, indicating that residues comprising the Vβ determinants of SEE are separate from residues that contribute to HLA-DR1 binding affinity. Computer models of the predicted structure of SEE revealed that the Vβ determinants of SEE are located on two adjacent solvent-exposed loops. Thus, the residues of SEE that mediate Vβ recognition may coalesce to form a TCR binding site with specificities for multiple TCR β-chains.