Abnormalities in CD4(+)CD25(+)Foxp3(+) regulatory T (T reg) cells have been implicated in susceptibility to allergic, autoimmune, and immunoinflammatory conditions. However, phenotypic and functional assessment of human T reg cells has been hampered by difficulty in distinguishing between CD25-expressing activated and regulatory T cells. Here, we show that expression of CD127, the alpha chain of the interleukin-7 receptor, allows an unambiguous flow cytometry-based distinction to be made between CD127(lo) T reg cells and CD127(hi) conventional T cells within the CD25(+)CD45RO(+)RA(-) effector/memory and CD45RA(+)RO(-) naive compartments in peripheral blood and lymph node. In healthy volunteers, peripheral blood CD25(+)CD127(lo) cells comprised 6.35 +/- 0.26% of CD4(+) T cells, of which 2.05 +/- 0.14% expressed the naive subset marker CD45RA. Expression of FoxP3 protein and the CD127(lo) phenotype were highly correlated within the CD4(+)CD25(+) population. Moreover, both effector/memory and naive CD25(+)CD127(lo) cells manifested suppressive activity in vitro, whereas CD25(+)CD127(hi) cells did not. Cell surface expression of CD127 therefore allows accurate estimation of T reg cell numbers and isolation of pure populations for in vitro studies and should contribute to our understanding of regulatory abnormalities in immunopathic diseases.