TNFR2 is predominantly expressed by a subset of human and mouse CD4 CD25FoxP3 T regulatory cells (Tregs). In this study, we characterized the phenotype and function of TNFR2 Tregs in peripheral lymphoid tissues of normal and tumor-bearing C57BL/6 mice. We found that TNFR2 was expressed on 30-40% of the Tregs of the peripheral activated/memory subset that were most highly suppressive. In contrast, TNFR2 Tregs exhibited the phenotype of naive cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4 CD25TNFR2 cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2 Tregs was considerably more potent than that of reportedly highly suppressive CD103 Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2 Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity that may account for tumor-infiltrating lymphocyte-mediated immune evasion by tumors. Copyright © 2008 by The American Association of Immunologists, Inc.