CD4+ CD25+ regulatory T (Treg) cells play a critical role in the maintenance of peripheral tolerance and the prevention of autoimmunity. In the present study, we have explored the characteristics of CD4+ CD25+ Treg cells in patients with rheumatoid arthritis (RA). The frequency and phenotype of CD4+ CD25+ T cells in paired samples of synovial fluid (SF) and peripheral blood (PB) from patients with RA and PB from normal controls were analysed. An increased frequency of CD4+ cells T cells expressing CD25 was detected in SF compared to PB from patients with RA. No significant difference was observed in the numbers of CD4+ CD25+ T cells in PB from patients and controls. SF CD4+ CD25+ T cells expressed high levels of CTLA-4 (both surface and intracellular), GITR and OX40, as well as Foxp3 transcripts. Functionally, SF CD4+ CD25+ T cells were impaired in their proliferative responses and could suppress the proliferation of their CD4+ CD25– counterparts. In conclusion, these data demonstrate that CD4+ CD25+ Treg cells, with the potential to regulate the function of effector T cells and antigen-presenting cells, accumulate in the synovium of patients with RA.