Background. Translational research in a relevant preclinical model is recommended before Treg-inducing protocols can be implemented in humans. We have characterized rhesus monkey CD25(+) cells phenotypically and functionally. Methods. The phenotype of CD4(+)CD25(high) cells was determined by FACS, focusing on established markers of mouse and human Treg cells. Percentages of cells positive for CD45RA, CD62L, and intracellular CTLA-4 and FOXP3 were compared between CD4(+)CD25(high) and CD4(+)CD25(-) cells. CD25(-) cells stimulated with anti-CD3, ConA, and/or allogeneic peripheral blood mononuclear cells were mixed with freshly isolated CD25(+) cells. The suppressive activity of the CD25(+) cells in vitro was assessed using several experimental conditions. Results. Rhesus monkey CD4(+)CD25(high) cells expressed high intracellular levels of CTLA-4 and FOXP3, whereas expression was negligible in CD4(+)CD25(-) cells. The CD25(high) population was mostly CD45RA(-), indicative of a memory phenotype. The CD25(+) cells were anergic, because they showed low proliferative responses, no interleukin-2 production, and some interferon-gamma and interleukin-10 production. Proliferation of CD4(+)CD25(-) cells stimulated by anti-CD3 or allogeneic cells was decreased when CD4(+)CD25(+) cells were added at a 1:1 ratio. In addition, we found that CD25(+) cells inhibited the interleukin-2 and interferon-gamma production by anti-CD3 -stimulated CD25(-) cells in a dose-dependent fashion, through a cell-cell contact-dependent mechanism. Conclusions. Rhesus monkey CD4(+)CD25(+) cells have similar phenotypic and functional characteristics as natural Tregs in humans. These findings allow testing of Treg expansion and induction protocols in a relevant preclinical model, the rhesus monkey.