Mechanisms of maintenance of transplantation tolerance induced in adult rats by depletive regimens of anti-CD4 before transplantation of vascularized heart allografts were studied. Despite the fact that there has been little evidence that tolerant lymphocytes could prevent allograft rejection after adoptive transfer, we demonstrated a suppressive role for lymphocytes from tolerant animals in vivo. These experiments analyzed the ability of lymphocytes from tolerant rats to protect passenger leukocyte-depleted Lewis heart grafts that had been "parked" in ACI rats (treated with pretransplant anti-CD4 and maintained for > 100 days) compared with their ability to protect transplantation of fresh Lewis heart grafts in naive ACI rats. Although parked Lewis heart grafts were rejected in unmanipulated ACI recipients, parked hearts (but not naive Lewis heart allografts), were permanently accepted by naive ACI rats when syngeneic tolerant spleen cells were adoptively transferred at the time of transplantation. Further, we demonstrated that the suppressor cells in the tolerant spleen cells were CD4+. These results suggest that CD4+ spleen cells from tolerant rats inhibit allograft recognition and may maintain allograft tolerance by blocking the indirect pathway of allorecognition.