The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2(bm12) cardiac allografts is restricted by CD4(+)CD25(+) regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2(bm12) cardiac allografts. In contrast to the long-term survival of B6.H-2(bm12) allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5(-/-) recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-gamma and IL-4 were markedly increased in spleens of B6.CCR5(-/-) versus wild-type recipients. Wild-type and B6.CCR5(-/-) mice had equivalent numbers of splenic FoxP3(+) Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro. However, diminished numbers of FoxP3(+) Tregs infiltrated B6.H-2(bm12) allografts in B6.CCR5(-/-) recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4(+)CD25(+) Tregs to CCR5(-/-) recipients restored long-term survival of B6.H-2(bm12) cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.