Human γδ T cells expressing the Vγ2Vδ2 antigen receptors recognize nonpeptide prenyl pyrophosphate and alkylamine antigens. We find that they also recognize staphylococcal enterotoxin A superantigens in a manner distinct from the recognition of nonpeptide antigens. Using chimeric and mutant toxins, SEA amino acid residues 20-27 were shown to be required for γδ TCR recognition of SEA. Residues at 200-207 that are critical for specific αβ TCR recognition of SEA do not affect γδ TCR recognition. SEA residues 20-27 are located in an area contiguous with the binding site of V β chains. This study defines a superantigen recognition site for a γδ T cell receptor and demonstrates the differences between Vγ2Vδ2+ T cell recognition of superantigens and nonpeptide antigens.