Naturally arising CD25⁺CD4⁺ regulatory T (T_R) cells can be exploited to establish immunologic tolerance to non-self antigens. In vivo exposure of CD25⁺CD4⁺ T cells from normal naive mice to alloantigen in a T cell-deficient environment elicited spontaneous expansion of alloantigen-specific CD25⁺CD4⁺ T_R cells, which suppressed allograft rejection mediated by subsequently transferred naive T cells, leading to long-term graft tolerance. The expanded T_R cells, which became CD25^low in the absence of other T cells, stably sustained suppressive activity, maintained expression levels of other T_R cell-associated molecules, including Foxp3, CTLA-4 and GITR, and could adoptively transfer tolerance to normal mice. Furthermore, specific removal of the T_R cells derived from originally transferred CD25⁺CD4⁺ T_R cells evoked graft rejection in the long-term tolerant mice, indicating that any T_R cells deriving from CD25⁻CD4⁺ naive T cells minimally contribute to graft tolerance and that natural T_R cells are unable to infectiously confer significant suppressive activity to other T cells. Similar antigen-specific expansion of T_R cells can also be achieved in vitro by stimulating naturally present CD25⁺CD4⁺ T cells with alloantigen in the presence of IL-2. The expanded CD25⁺CD4⁺ T cells potently suppressed even secondary MLR in vitro and, by in vivo transfer, established antigen-specific long-term graft tolerance. Thus, in vivo or in vitro, direct or indirect ways of antigen-specific expansion of naturally arising Foxp3⁺CD25⁺CD4⁺ T_R cells can establish antigen-specific dominant tolerance to non-self antigens, and would also be instrumental in re-establishing self-tolerance in autoimmune disease and antigen-specific negative control of pathological immune responses.