Dominant tolerance is mediated by regulatory T cells (T reg) that control harmful autoimmune T cells in the periphery. In this study, we investigate the implication of T reg in modulating infiltrating T lymphocytes in human metastatic melanoma. We found that CD4+ CD25 high T cells are overrepresented in metastatic lymph nodes (LNs) with a 2-fold increased frequency compared with both tumor-free LNs and autologous PBMCs. These cells express the Foxp3 transcription factor, display an activated phenotype, and display a polyclonal TCR Vβ chain repertoire. They inhibit in vitro the proliferation and cytokine production of infiltrating CD4+ CD25− and CD8+ T cells (IL-2, IFN-γ) through a cell-contact-dependent mechanism, thus behaving as T reg. In some cases, the presence of T reg type 1/Th3-like lymphocytes could also be demonstrated. Thus, T reg are a major component of the immunosuppressive microenvironment of metastatic melanoma LNs. This could explain the poor clinical response of cancer patients under immunotherapeutic protocols, and provides a new basis for future immunotherapeutic strategies counteracting in vivo T reg to reinforce local antitumor immune responses.