CD4(+)CD25(+) regulatory T cells have been shown to regulate a variety of autoimmune and allogeneic responses in mice and humans. The role of CD4(+)CD25(+) cells in regulating alloresponses in human transplant recipients remains uncertain. Previous research has demonstrated a reduced frequency of direct pathway donor-specific T cells in renal transplant recipients when compared with the frequency of T cells reactive to an HLA-matched third party. A number of mechanisms have been proposed to account for this finding; the purpose of this study was to determine whether CD4(+)CD25(+) cells play a significant role. Twelve stable renal transplant patients were investigated using limiting dilution assay (LDA) and ELISPOT for interferon-gamma to determine the effect of depleting CD4(+)CD25(+) cells on the direct pathway alloresponse. The percentage of CD4(+)CD25(+) cells in the peripheral blood of the study patients was equivalent to that of healthy controls. Furthermore, in no case did depletion of CD4(+)CD25(+) cells significantly increase the frequency of donor-specific T cells detected by LDA. This was also found with ELISPOT in all except one patient, in whom depletion revealed an increased frequency of alloreactive T cell to both donor and third party. Finally, kinetic analysis of the LDA data did not indicate regulation against donor when compared with third party. It is concluded that the action of CD4(+)CD25(+) regulatory cells is not the main mechanism of donor-specific hyporesponsiveness in the direct pathway of allorecognition.