Superantigens encoded in the genome or released by bacteria have been identified as potent modulators of the murine immune system. High frequencies of mature or immature T cells are activated or intrathymically deleted when superantigens cross-link MHC class II molecules and the V beta element of the TCR. The V beta specificity discriminates superantigens from polyclonal T cell stimulators as well as specific Ag and determines the immunomodulatory role in shaping the T cell repertoire. A similar regulatory function of superantigens in the human immune system is less well established. Here, we have studied a series of human T cell clones sharing the TCR V beta 6 element and describe a surprising heterogeneity in their responsiveness to staphylococcal exotoxins. The V beta 6 gene segment had the ability to respond to all staphylococcal enterotoxins (SE); however, for individual T cell clones, there was a clear predominance of SE C3 reactivity compared to SE B and SE C2. The clonal heterogeneity of SE responsiveness did not correlate to sequence polymorphisms in the fourth hypervariable region of the V beta 6 segment, the presumptive binding site for superantigens. Superantigen reactivity was crucially influenced by the presenting HLA-DR molecule, especially when the superantigen served as a coligand, enhancing or suppressing the Ag-specific activation of the TCR. These data suggest that the correlation between human TCR V beta gene segments and superantigen responses is not stringent. Potential intrathymic deletion mechanisms controlled by superantigens may be less selective in humans and may result in a leakiness influenced by the host HLA-DR molecules.