Previously, we found that co-expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3 cells present in CD25, CD25 and even CD25 subsets of CD4 cells expressed high levels of TNFR2. Consequently, TNFR2-expressing CD4 CD25 Treg included all of the FOXP3 cells present in the CD4CD25 subset as well as a substantial proportion of the FOXP3 cells present in the CD4 CD25 subset. Flow cytometric analysis of PB identified five-fold more Treg, determined by FOXP3 expression, in the CD4 CD25TNFR2 subset than in the CD4 CD25 subset. In addition, similar levels of FOXP3 cells were identified in both the CD4CD25 TNFR2 and CD4CD25 CD127 subsets. Furthermore, the CD4CD25 TNFR2 subset expressed high levels of CTLA-4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4CD25TNFR2 cells were anergic and markedly inhibited the proliferation and cytokine production of co-cultured T-responder cells. In contrast, CD4 CD25TNFR2 and CD4CD25 TNFR2 T cells did not show inhibitory activity. As some non-Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population thatmay prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.