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 α 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.