CD4 regulatory T cells (Tregs) can be subdivided into two subsets according to Ly-6C expression in the periphery. Phenotypic analysis, imaging, and adoptive-transfer experiments of peripheral Ly-6C− and Ly-6C+ Tregs reveal that the nonexpression of Ly-6C by∼ 70% of peripheral Tregs depends on TCR signaling events. Interestingly, Ly-6C− Tregs express higher surface amounts of key immunosuppressive molecules than do Ly-6C+ Tregs and produce constitutively anti-inflammatory cytokines. In line with their phenotype, Ly-6C+ Tregs exhibit poor suppressive capacities in vitro and in vivo. Finally, although Ly-6C− Tregs maintain their numbers with age, Ly-6C+ Tregs gradually disappear. Altogether, our data strongly suggest that both the survival and suppressive functions of peripheral CD4 Tregs rely on their ability to receive strong TCR signals.