CD4⁺CD25⁺ regulatory T (T_reg) cells are pivotal for the maintenance of self-tolerance, and their adoptive transfer gives protection 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 phenotypic and functional characteristics similar to those of murine CD4⁺CD25⁺ T_reg cells: namely, hyporesponsiveness to T-cell receptor (TCR) stimulation and suppression of CD25^- T cells. Thus far, the detailed characterization and potential clinical application of human CD4⁺CD25⁺ T_reg cells have been 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 antigen-presenting cells for repeated stimulation via CD3 and CD28 in the presence of high-dose interleukin 2 (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 maintained expression of the lymph node homing receptors L-selectin (CD62L) and CCR7. The ability to rapidly expand human CD4⁺CD25^high T_reg cells on a large scale will not only facilitate their further exploration but also accelerate their potential clinical application in T cell–mediated diseases and transplantation medicine.