The balanced manifestation of effector functions and the generation of long-living memory cells is a hallmark of efficient CD8(+) T-cell response. Accumulating data pinpoint CD4(+) CD25(high) regulatory T (Treg) cells as a key factor for the inefficiency of CD8(+) T-cell responses in viral persistence. Little is known about the effects of Treg cells on the homeostasis of healthy donor CD8(+) T cells. The present study demonstrates that Treg cells exert differential effects on CD8(+) T-cell subsets. Treg cells inhibited mostly the polyclonal proliferation of CD27(-) effector cells compared with CD27(+) memory CD8(+) T cells. Moreover, they inhibited the polyclonal and antigen-driven differentiation of memory cells into functional effectors as defined by IFN-gamma secretion and induction of CD160 expression. Finally, Treg cells reduced the apoptosis of memory but not of effector and terminal effector cell populations. These effects were at least in part mediated by a decreased expression of PD-L1, but not of programmed death 1 (PD-1), on CD8(+) T cells after activation. Thus, in the setting of a healthy immune system, Treg cells fine-tune the memory/effector cell balance and promote the accumulation of long-living memory cells in case of strong stimulation.