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.