Staphylococcal enterotoxin H (SEH) is a bacterial superantigen secreted by Staphylococcus aureus. Superantigens are presented on the MHC class II and activate large amounts of T cells by cross-linking APC and T cells. In this study, RT-PCR was used to show that SEH stimulates human T cells via the Valpha domain of TCR, in particular Valpha10 (TRAV27), while no TCR Vbeta-specific expansion was seen. This is in sharp contrast to all other studied bacterial superantigens, which are highly specific for TCR Vbeta. It was further confirmed by flow cytometry that SEH stimulation does not alter the levels of certain TCR Vbeta. In a functional assay addressing cross-reactivity, Vbeta binding superantigens were found to form one group, whereas SEH has different properties that fit well with Valpha reactivity. As SEH binds on top of MHC class II, an interaction between MHC and TCR upon SEH binding is not likely. This concludes that the specific expansion of TCR Valpha is not due to contacts between MHC and TCR, instead we suggest that SEH directly interacts with the TCR Valpha domain.