CD4+CD25+ regulatory T cells (T(regs)) have been shown to inhibit graft-versus-host disease (GVHD) in murine models, and this suppression was mediated by T(regs) expressing the lymphoid homing molecule l-selectin. Here, we demonstrate that T(regs) lacking expression of the chemokine receptor CCR5 were far less effective in preventing lethality from GVHD. Survival of irradiated recipient animals given transplants supplemented with CCR5-/- T(regs) was significantly decreased, and GVHD scores were enhanced compared with animals receiving wild-type (WT) T(regs). CCR5-/- T(regs) were functional in suppressing T-cell proliferation in vitro and ex vivo. However, although the accumulation of T(regs) within lymphoid tissues during the first week after transplantation was not dependent on CCR5, the lack of function of CCR5-/- T(regs) correlated with impaired accumulation of these cells in the liver, lung, spleen, and mesenteric lymph node, more than one week after transplantation. These data are the first to definitively demonstrate a requirement for CCR5 in T(reg) function, and indicate that in addition to their previously defined role in inhibiting effector T-cell expansion in lymphoid tissues during GVHD, later recruitment of T(regs) to both lymphoid tissues and GVHD target organs is important in their ability to prolong survival after allogeneic bone marrow transplantation.