Current therapies in transplantation require continuous immunosuppression and do not result in transplantation tolerance. It is increasingly appreciated that CD4(+)CD25(+) regulatory T-cell (T(REG)) activation is pivotal for the induction and maintenance of peripheral tolerance. To optimally exploit T(REG) in allograft tolerance, we investigated how to further harness their function. In vitro, CD4(+)CD25(+)T cells were expanded by allogeneic bone-marrow derived DC or polyclonal stimulation and were compared in suppressive capacity and phenotype. In vivo, naive allogeneic CD4(+)CD25(+)T cells were analyzed in wild type hosts for proliferative capacity and suppressive capacity upon priming by alloantigen. DC of donor origin were found to potently stimulate alloreactive T(REG)in vitro. This was accompanied by a substantial enhancement of the suppressive capacity of the T(REG) population as a whole, likely due to a proportional rise of alloreactive T(REG) as indicated by CFSE analysis. In vivo analysis of infused naturally occurring allogeneic T(REG) revealed a robust proliferative capacity for T(REG) upon stimulation. Moreover, allogeneic skin transplantation resulted in enhanced capacity of the T(REG) population to suppress the response towards donor antigens. Combining, activation of alloreactive T(REG) is an intrinsic part of the regular alloimmune response and this feature can be exploited for therapeutic purposes. We propose that selectively favoring the effects of alloreactive T(REG) is a pivotal element in inducing graft acceptance.