CD4+ CD25+ high T cells are potent regulators of autoreactive T cells. However, it is unclear how regulatory CD4+ CD25+ high cells discriminate between desirable inflammatory immune responses to microbial Ags and potentially pathologic responses by autoreactive T cells. In this study, an in vitro model was created that allowed differential activation of regulatory CD4+ CD25+ high and responder CD4+ T cells. If CD4+ CD25+ high regulatory cells were strongly activated, they maintained suppressive effector function for only 15 h, while stimulation with weaker TCR stimuli produced regulatory cells that were suppressive until 60 h after activation. In contrast, strongly activated CD4+ responder T cells were resistant to regulation at all time points, while weakly stimulated CD4+ cells were sensitive to suppression until 38 or 60 h after activation depending upon the strength of the stimulus. The extent of suppression mediated by CD4+ CD25+ high cells also depended on the strength of stimulation in an Ag-specific system. Thus, the stronger the TCR signal, the more rapidly and more completely the responder cells become refractory to suppression.