Donor-specific heart allograft acceptance can be induced in the MHC-mismatched LEW.1 W to LEW.1A rat by donor-specific transfusions. Whereas the induction phase of tolerance has been studied in detail, its maintenance remained poorly understood. Here, we performed a side-by-side comparison of CD25+ and CD25- splenic T cells of 100-day tolerant rats. Administration of CD25- T cells from tolerant rats to sublethally irradiated recipients transferred long-term graft survival. These CD25- T cells displayed a decreased donor-specific response in the mixed lymphocyte reaction and presented suppressive activity. These CD25- T cells accumulated IFN-gamma, IL-10 and Foxp3 transcripts. The in vitro suppressive activity of CD25- T cells required both cell contact and soluble factors (IL-10 and IFN-gamma). The CD25+ T cells from tolerant rats did not show any modification of their regulatory properties. We show that splenic CD25- T cells of tolerant rats contribute to the maintenance of tolerance following the transplantation. Our data show that regulatory T cells are not restricted to the CD4+ CD25+ T cell subset and provide new insights on the mechanisms of tolerance to allograft following donor cell priming.