CD4⁺ CD25⁺ regulatory T (T_reg) 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⁺ T_reg 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⁺ T_reg cells, with the potential to regulate the function of effector T cells and antigen-presenting cells, accumulate in the synovium of patients with RA.