BACKGROUND: Natural regulatory T cells (nTreg) can suppress different immune-cell responses and maintain the balance between tolerance and immunity in the individual. These cells are defined by CD4+ , CD25hi, and FOXP3+ expression, although a variety of other nTreg-associated markers have been reported to be expressed at different levels (e.g., HLA-DR, CTLA-4, GARP, Helios, CD39, etc.), presumably reflecting different functional stages of the heterogeneous nTreg population. Several markers show low/negative expression (i.e., CD127, CD49d, and CD26), but none of these markers are specific to nTreg. CD25hi expression has been a useful surface marker to identify/isolate nTreg; however, CD25 is also expressed on “adaptive” or “induced” Treg, as well as in activated conventional T cells. In addition, the fact that FOXP3 is also found in CD25 low/negative CD4+ T cells, and in a subset of CD8+ T cells, further complicates the definition of a specific nTreg marker. Although CD4+, CD25hi, and CD127low/negative markers characterize the majority of nTreg, it is still imperative to find additional surface-marker combinations that improve the identification/isolation of nTreg and their subsets. Herein, we present evidence that CD4+ CD25hi CD6(lo/-) nTreg have high expression of FOXP3and exhibit in vitro suppressive activity on CD8+ T-cell proliferation. Dot-plot analyses of CD4+ cells, with CD6, CD127, CD49d, or CD26 reveal that a higher enrichment yield of CD25hi FOXP3+ cells can be achieved in the combined CD6(lo/-) CD127(lo/-) population. We conclude that FOXP3+ nTreg cells are characterized by CD6(lo/-) expression, providing a new tool for the identification of nTreg cells without recourse to intracellular staining, and for the purification of these cells by negative selection.