Donor-specific hyporesponsiveness as occurs after allogeneic kidney transplantation may be mediated by repression of effector cells by a specific subset of T-cells: the CD4(+) CD25(bright+) FoxP3(+) regulatory T-cells (Tregs). Here, we examined the suppressive capacity of Tregs isolated from the leukafereses product of 6 kidney transplant recipients, by reconstituting Tregs to responder T-cells at several time-points after initiation of proliferation. We show that Tregs derived from kidney transplant patients potently restrain proliferation to donor-antigens and 3rd party-antigens in classic reconstitution assays (i.e. addition of Tregs at the start of the co-incubation). However, when Tregs were added 5 days after initiation of proliferation, they were still capable of suppressing proliferation to donor-antigens (by 38%) but no longer to 3rd party-antigens. Thus, we conclude that the potency of Tregs to suppress reactivity to specific antigens should be determined by reconstitution to ongoing reactions.