To elucidate the parameters that lead to superantigen induced non-responsiveness, an in vitro model for studying primary and secondary responses to the bacterial superantigen staphylococcal enterotoxin B (SEB) was established. Upon re-activation with SEB, in vitro SEB primed T cells show an early proliferative response that 'quenches' in time and is severely impaired 3 days after re-stimulation. Despite their overall impaired proliferative capacity and IL-2 production, these T cells are able to produce IFN-gamma and to up-regulate activation markers CD69 and IL-2R alpha upon re-stimulation with SEB, demonstrating that SEB non-responsiveness is not absolute. Rather, it reflects the inability to mount an ongoing proliferative response upon re-stimulation with SEB. Our results also demonstrate that SEB-induced non-responsiveness is not simply the result of presentation in the absence of co-stimulation, since presentation of SEB on highly purified dendritic cells during the primary response did not prevent the induction of non-responsiveness. As previously shown, SEB induces a Th1 phenotype in responding CD4+ T cells. Skewing towards a Th2 phenotype by adding IL-4 and antibodies to IFN-gamma did not prevent the induction of non-responsiveness by SEB. Interestingly, T cells pretreated with plate-bound anti-CD3 epsilon and anti-V beta 8 were also non-responsive to SEB re-stimulation. Thus, non-responsiveness to SEB (defined here as inability to produce IL-2 and proliferate) seems to reflect an intrinsic inability of previously activated T cells to respond to SEB, probably reflecting differences in signal transduction pathways used by naive versus previously activated T cells.