Although transplantation has become a mainstay therapy for end-stage organ failure, clinical success continues to be limited by the ongoing need for nonspecific immunosuppressive therapy that reduces the risk of graft rejection but also brings with it unwanted effects, such as increased susceptibility to infection and malignancy together with failure to prevent chronic rejection. The aim of this study was to investigate the role of CD25+CD4+T cells in controlling responses to donor allo-antigens. Healthy volunteers peripheral blood was used to establish the assays. The proliferative response of enriched CD4+T cells in the presence and absence of CD25+CD4+ T cells was compared using two methods, tritiated thymidine incorporation, which provide an estimate of the total number of dividing cells in mixed lymphocyte reaction, and labelling of the responding population with CFSE, a fluorescent dye, that stains intracellular proteins, and partitioned equally between daughter cells at each cell division. Removal of CD25+ cells resulted in dramatic proliferation that could be detected using both methods. The same techniques were applied to analyse PBMCS before and after depletion of CD25+ cells. Interestingly, a dramatic proliferation of both CD4+T cells and CD8+T cells was observed. Depletion of CD25+T cells resulted in dramatic Cytotoxic response through increase of the expression of intra-cellular INF-_ in CD4+T cells and dramatic increase of the intracellular Granzyme B molecules in CD8+T cells. Our results prove the potential regulatory role of CD25+CD4+T cells in controlling responses to donor allo-antigens which may account for long term graft survival.