Donor derived CD4+CD25+ regulatory T (Treg) cells do not induce graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but actively suppress GVHD induced by CD25− conventional T (Tconv) cells when co-transplanted at a 1:1 ratio in murine model systems. Purified human CD4+CD25+ Treg cells display similar phenotypic and functional characteristics as their murine counterparts, i.e. they constitutively express high levels of intracellular CTLA-4, preferentially express the forkhead/winged helix transcription factor Foxp3, they are anergic to T cell receptor-mediated stimulation, and suppress the proliferation of Tconv cells after polyclonal or allogeneic activation in vitro. Thus, their adoptive transfer is a promising strategy for the prevention of GVHD in patients after allogeneic BMT. For potential clinical applications, efficient protocols for the isolation of CD4+CD25+ Treg cells from peripheral blood are required. In laboratory-scale experiments we determined that depletion of B cells (that partially express CD25), followed by repetitive positive selection of the remaining CD25+ cells with paramagnetic beads, yields a more than 90% pure population of CD4+CD25+ T cells. We now applied this protocol for the large-scale isolation of human CD4+CD25+ T cells from leukapheresis products of healthy volunteers under GMP conditions using the CliniMACS® device. B cell depletion with CD19-beads, followed by three cycles of CD25-enrichment with CD25-coupled microbeads resulted in an average 90% purity of CD4+CD25+ T cells (range 86% to 94%). Positively selected cells constituted on average 1.56% of MNC (range 0.84% to 2.0%), resulting in absolute numbers of 63 to 261 x 106 cells, depending on the initial size of the leukapheresis product. Since T cells with regulatory capacity predominantly reside within the CD4+CD25high subpopulation, we further determined the relative proportion of CD25high to CD25intermediate CD4+ T cells within the cell product. CD4+CD25high T cells represented on average 42% of the purified CD4+CD25+ T cell population (range 26% to 60%) as compared to 10.6% (range 8.5% to 13%) of the CD4+CD25+ T cells within the starting population. CD4+CD25+ T cells isolated according to this GMP protocol expressed higher levels of Foxp3 mRNA and Foxp3 protein as well as higher levels of intracellular CTLA-4 as compared to CD4+CD25− T cells. In addition, they showed functional characteristics of Treg cells as they were hypoproliferative to polyclonal stimulation in vitro and inhibited the proliferation of co-cultured CD4+CD25− T cells in a standard suppression assay. The ability to isolate highly enriched Treg cell populations in sufficient numbers under GMP conditions will accelerate their clinical application in allogeneic BMT.