Thymus-derived CD4⁺CD25⁺ regulatory T cells suppress autoreactive CD4⁺ and CD8⁺ T cells and thereby protect from autoimmunity. In animal models, adoptive transfer of CD4⁺CD25⁺ regulatory T cells has been shown to prevent and even cure autoimmune diseases as well as pathogenic alloresponses after solid organ and stem-cell transplantations. We recently described methods for the efficient in vitro expansion of human regulatory T cells for clinical applications. We now demonstrate that only CCR7- and L-selectin (CD62L)–coexpressing cells within expanded CD4⁺CD25^high T cells maintain phenotypic and functional characteristics of regulatory T cells. Further analysis revealed that these cells originate from CD45RA⁺ naive cells within the CD4⁺CD25^high T-cell compartment, as only this subpopulation homogeneously expressed CD62L, CCR7, cytotoxic T lymphocyte–associated antigen-4 (CTLA-4), and forkhead box P3 (FOXP3), produced no inflammatory cytokines and maintained robust suppressive activity after expansion. In contrast, cell lines derived from CD45RA^– memory-type CD4⁺CD25^high T cells lost expression of lymph node homing receptors CCR7 and CD62L, contained interleukin-2 (IL-2) and interferon-γ (IFN-γ) as well as IL-10–secreting cells, showed only moderate suppression and, most importantly, did not maintain FOXP3 expression. Based on these unexpected findings, we suggest that isolation and expansion of CD45RA⁺ naive CD4⁺ CD25^high T cells is the best strategy for adoptive regulatory T (Treg)–cell therapies.