CD4⁺ regulatory T cells (T_reg) play an indispensable role in tolerance to peripheral antigens, but the origin of the T_reg pool in the adult remains unclear. Thus, while thymic commitment of T_reg has been demonstrated, evidence also exists for the peripheral recruitment of naïve tissue‐specific T cells into T_reg functions. Anti‐myelin basic protein TCR transgenic mice spontaneously develop autoimmune encephalomyelitis when "monoclonal", but are protected by adoptive transfer of CD4⁺ cells from wild‐type donors. We have now used this transfer system to investigate whether previously infused T_reg can recruit transgenic T cells to regulatory functions. The results show that transgenic T cells from protected animals did not transfer tolerance to secondary recipients, and that elimination of donor T_reg in protected recipients resulted in rapid onset of disease. In addition, T_reg‐containing T cell susbsets were highly enriched for proliferating cells in vivo, which was also the case for CD4⁺CD25⁺ T cells in normal animals. These observations thus exclude peripheral differentiation of T_reg in this particular system, and indicate that expansion of thymically committed cells ensures the maintenance of the peripheral T_reg pool in the adult.