Rapamycin is an immunosuppressive compound that is currently used to prevent acute graft rejection in humans. In addition, rapamycin has been shown to allow operational tolerance in murine models. However, a direct effect of rapamycin on T regulatory (Tr) cells, which play a key role in induction and maintenance of peripheral tolerance, has not been demonstrated so far. Here, we provide new evidence that rapamycin selectively expands the murine naturally occurring CD4⁺CD25⁺FoxP3⁺ Tr cells in vitro. These expanded Tr cells suppress proliferation of syngeneic T cells in vitro and prevent allograft rejection in vivo. Interestingly, rapamycin does not block activation-induced cell death and proliferation of CD4⁺ T cells in vitro. Based on this new mode of action, rapamycin can be used to expand CD4⁺CD25⁺FoxP3⁺ Tr cells for ex vivo cellular therapy in T-cell-mediated diseases. (Blood. 2005;105:4743-4748)