Naturally arising CD4+CD25+ regulatory T (Treg) cells can be exploited to establish immunologic tolerance to allogeneic transplants. In vivo exposure of CD4+CD25+ T cells from normal naive mice to alloantigen in a T cell-deficient environment elicits spontaneous expansion of alloantigen-specific CD4+CD25+ natural Treg cells, which are able to suppress allograft rejection mediated by subsequently transferred naive T cells, leading to long-term graft tolerance. Similar antigen-specific expansion of natural Treg cells can also be achieved in vitro by stimulating CD4+CD25+ T cells from normal animals with alloantigen in the presence of high doses of interleukin-2. The expanded CD4+CD25+ Treg cells are even capable of suppressing secondary mixed leukocyte reaction in vitro and, by in vivo transfer, establishing antigen-specific long-term graft tolerance. Thus, in vivo or in vitro, direct or indirect ways of alloantigen-specific expansion of naturally arising CD4+CD25+ Treg cells can establish antigen-specific dominant tolerance to allogeneic transplants.