CD4⁺CD25⁺ regulatory T (T_reg) cells are pivotal for the maintenance of self-tolerance and their adoptive transfer protects from autoimmune diseases and pathogenic alloresponses after solid organ or bone marrow transplantation in murine model systems. In vitro, human CD4⁺CD25⁺ T_reg cells display similar phenotypic and functional characteristics as murine CD4⁺CD25⁺ T_reg cells, namely hyporesponsiveness to TCR stimulation and suppression of CD25^- T cells. Thus far, the detailed characterization and potential clinical application of human CD4⁺CD25⁺ T_reg cells was hampered by their paucity in peripheral blood and the lack of appropriate expansion protocols. Here we describe the up to 40,000-fold expansion of highly purified human CD4⁺CD25^high T cells in vitro through the use of artificial APC for repeated stimulation via CD3 and CD28 in the presence of high dose IL-2. Expanded CD4⁺CD25^high T cells were polyclonal, maintained their phenotype, exceeded the suppressive activity of freshly isolated CD4⁺CD25^high T cells and showed characteristics of central memory T cells. The ability to rapidly expand human CD4⁺CD25^high T_reg cells large scale will not only facilitate their further exploration but also accelerate their potential clinical application in T cell-mediated diseases and transplantation medicine.