CD25^highCD4⁺ T cells potential role in maintaining self tolerance has been demonstrated in healthy human subjects. Their role in long term renal graft transplant patients is still unclear. The aim was to evaluate the ability of CD25^highCD4⁺ T cells to regulate responses to donor allo-antigens in clinically stable renal transplant patients. Peripheral blood samples from a cohort of 30 living related renal transplant recipients were studied. Group A, included 15 rejection free transplant recipients with stable graft function. Group B, included 15 transplant recipients suffering from chronic graft dysfunction. The proliferative responses of CD4⁺ and CD8⁺ T cells in the presence and absence of CD25^highCD4⁺ T cells was assessed by targeting the loss of CFSE staining from dividing cells in mixed lymphocyte co-cultures. Flow cytometry Phenotyping revealed a higher absolute number of CD25^highCD4⁺ cells in Group A as compared with Group B (p=0.019) with difference to those detected in healthy volunteers (P=0.084). In CFSE_MLR assay, depletion of CD25^highCD4⁺ in rejection free patients (group A) samples, showed active regulation in 11 (74%) of 15 assays to donor stimulatory cells but not third party control. In chronic rejection patients (group B), depletion of CD25^highCD4⁺ failed to show any regulation response in all of 15 assays. CFSE assay enabled a detailed evaluation of the regulatory function of CD25^high T cells in long term renal transplant recipients. CD25^high T cells in the peripheral blood of renal transplant recipients’ mediated specific regulation towards donor alloantigens and not the third party controls.