Little is known about the in vivo conditions in which CD4+ CD25+ regulatory T cells (T reg) exert their suppressive effect in nonlymphopenic mice. To this end, we analyzed T reg-mediated suppression of expansion and cytokine production at different levels of Ag-specific CD4+ CD25− T cell activation. Using Ab-mediated depletion of endogenous T reg, we show that basal immunosuppression is dependent on effector T cell activation. These polyclonal T reg, which were poorly activated in our immunization conditions, were effective in weak but not high T cell activation context. In contrast, the same immunization conditions led to proliferation of cotransferred Ag-specific T reg. Those efficiently inhibited T cell proliferation and cytokine production even in strong T cell activation context. Interestingly, T reg selectively suppressed expansion or cytokine production depending on the experimental approach. The importance of the immune context for efficient suppression is further supported by the observation that T reg depletion exacerbated diabetes of NOD mice only at the early stage of the disease. Overall, our study suggests that T reg-mediated suppression depends on the relative activation of T reg and effector T cells in vivo. This balance may be a critical factor in the regulation of immune responses.