We previously demonstrated the presence of regulatory T cells (Tregs) in lymph nodes (LNs) from rats made tolerant to a kidney allograft by donor peripheral blood mononuclear cell (PBMC) infusion. Here, we investigated the origin of Treg and characterized their phenotype and mechanisms underlying their suppressive effect. At different points after PBMC infusion, thymus, LN, and graft-infiltrating -lymphocyte's (GIL) alloreactivity was evaluated in mixed lymphocyte reaction (MLR), coculture, and transwell experiments. GIL phenotype (by fluorescence-activated cell sorting and immunohistochemistry) and cytokines mRNA expression were analyzed. Before transplantation, CD4(+) thymocytes and LN cells from donor PBMC-infused rats showed a reduced anti-donor but a normal anti-third-party proliferation. Anti-donor hyporesponsiveness was reverted by interleukin (IL)-2. CD4(+) thymocytes had no regulatory activity on a naïve MLR. Treg appeared in LN at 60 days post-transplant. CD4(+)-GIL isolated early (5 days) and late post-transplant (days 60-80) were hyporesponsive and suppressed a naïve MLR. IL-10 mRNA was upregulated in GIL and an anti-IL-10 monoclonal antibody reverted their inhibitory effect. Cell-to-cell contact potentiated the suppressive activity of CD4(+)-GIL. We suppose that allograft tolerance in this model is mediated by pretransplant generation of anergic cells in the thymus, which may have a permissive role to prevent early graft disruption. The healed graft is a source of donor antigens, which led to early selection of Treg. In the late phase, tolerance is maintained by appearance of Treg in LN.