Although regulatory T (T reg) cells are thought to develop primarily in the thymus, the peripheral events that shape the protective T reg cell population are unclear. We analyzed the peripheral CD4 + T cell receptor (TCR) repertoire by cellular phenotype and location in mice with a fi xed TCR ␤ chain. We found that T reg (Foxp3 + ) cells showed a marked skewing of TCR usage by anatomical location in a manner similar to antigen-experienced (CD44 hi Foxp3 -) but not naive (CD44 lo Foxp3 -) cells, even though CD44 hi and T reg cells used mostly dissimilar TCRs. This was likely unrelated to peripheral conversion, which we estimate generates only a small percentage of peripheral T reg cells in adults. Conversion was readily observed, however, during the immune response induced by Foxp3 -cells in lymphopenic hosts. Interestingly, the converted Foxp3 + and expanded Foxp3 -TCR repertoires were different, suggesting that generation of Foxp3 + cells is not an automatic process upon antigen activation of Foxp3 -T cells. Retroviral expression of these TCRs in primary monoclonal T cells confi rmed that conversion did not require prior cellular conditioning. Thus, these data demonstrate that TCR specifi city plays a crucial role in the process of peripheral conversion and in shaping the peripheral T reg cell population to the local antigenic landscape.