OBJECTIVE Previous studies have shown that the suppressive capacity of CD4+,CD25++ T cells is compromised in patients with rheumatoid arthritis (RA) and restored by anti-tumor necrosis factor alpha (anti-TNFalpha) therapy. Given the lack of specific cell surface markers for human Treg cells, this study aimed to define surface markers for identifying and enriching Treg cells with enhanced regulatory ability within the CD4+,CD25++ T cell compartment and to provide additional understanding of the effects of anti-TNFalpha antibodies in humans. METHODS The expression of membrane-bound TNFalpha in human peripheral blood CD4+ T cells was analyzed by flow cytometry in healthy individuals and RA patients before and after anti-TNFalpha treatment. Membrane-bound TNFalpha-positive and TNFalpha-negative CD4+,CD25++ T cells were purified by fluorescence-activated cell sorting, and their suppressive capacity was assessed in vitro by a standard suppression assay. RESULTS A substantial number of CD4+,CD25++ T cells expressed membrane-bound TNFalpha. Membrane-bound TNFalpha-positive CD4+,CD25++ T cells displayed reduced antiinflammatory cytokine production and less potent suppressor capacity, since 4 times more cells were required to achieve 50% inhibition compared with their membrane-bound TNFalpha-negative counterparts. Treatment of RA patients with TNFalpha-specific antibodies led to a reduction in the number of membrane-bound TNFalpha-positive CD4+,CD25++ T cells from peripheral blood. CONCLUSIONS Our data indicate that the absence of membrane-bound TNFalpha on CD4+,CD25++ T cells can be used to characterize and enrich for Treg cells with maximal suppressor potency. Enrichment of membrane-bound TNFalpha-negative CD4+,CD25+ cells in the CD4+,CD25++ T cell compartment may contribute to restoring the compromised suppressive ability of CD4+,CD25++ T cell populations in RA patients after anti-TNFalpha treatment.