TNFR2 is predominantly expressed by a subset of human and mouse CD4(+)CD25A(+)FoxP3(+) 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 bad minimal suppressive activity. Although not typically considered to be Tregs, CD4(+) CD25(-) TNFR2(+) 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.