Transcription of CD69 starts 30 min after the T cell face antigenic stimulation. CD69 as an early activation marker found to correlate with the results of mixed lymphocytic reaction. The potential role of CD25+CD4+ T cells in maintaining self tolerance has been proved in healthy human subjects. Their existence and role in long term renal transplant graft acceptor recipients still unclear. The aim of this study was to develop ex-vivo assay to evaluate the ability of CD25+CD4+T cells to control responses to donor allo-antigens in long term transplant recipients. The proliferative responses of CD4+T cells in the presence and absence of CD25+CD4+ T cells was assessed using two methods. First, tritiated thymidine incorporation into dividing cells, which provides an estimate of the total number of dividing cells in mixed lymphocyte reaction, and second, labeling of the responding population with CFSE, a fluorescent dye, that stains intracellular proteins, and partitioned equally between daughter cells at each cell division. A cohort of 20 long term renal graft survivors (>5 years) was studied, 7 recipients (35%) demonstrated significant high levels of CD25 (IL2 _ receptor) expression (P=0.0117) compared to their donors. Depletion of CD25+T cells resulted in dramatic proliferation of both CD4+T cells and CD8+T cells in response to donor antigens (P= < 0.001) but not to third party control. From our work, it is clear that CD25+CD4+ T cells mediate antigen specific suppression to donor allo-antigens. Not all transplant recipients able to shift to tolerant state. To our knowledge, this is the first time to apply CFSE assay to analyze human CD25 T cells. CFSE assay offered an advantage of additional information about the behavior of defined leukocyte subpopulations.