Regulatory CD4+ T cells (Treg) are essential to maintain self-tolerance. Release of natural Treg from the thymus is believed to commence soon after birth, but it is unclear how many are produced by "conversion" in the periphery, whether numbers are maintained after puberty by general homeostatic mechanisms that regulate lymphocyte numbers, or whether significant numbers are produced by the involuted thymus. To address the origin of Treg in normal adult rats, we focused on recent thymus emigrants (RTE). Approximately 30% of CD4+CD25+forkhead box p3 (Foxp3)+ Treg expressed markers associated with RTE. Following thymectomy, numbers of cells expressing these markers fell by 80% within 30 days. Furthermore, although only approximately 5% of CD4+ single-positive thymocytes expressed Foxp3 within 24 h after intrathymic injection of FITC, more than 30% of the labeled CD4+ RTE were Foxp3+, suggesting that some RTE may acquire Foxp3 in the periphery. Thus, some RTE may acquire Foxp3 rapidly after emigration from the thymus. Treg are dividing rapidly with apparent half-lives of approximately 18 days and approximately 7 days for the CD4+CD25+Foxp3+ and CD4+CD25-Foxp3+ subsets, respectively. The apparently slower turnover of CD4+CD25+Foxp3+ cells is a result of CD4+CD25+Foxp3+ --> CD4+CD25-Foxp3+ conversion, with no loss of regulatory function. Taken together, the data suggest that Treg in adults are relatively short-lived and that their numbers are maintained by rapid cell division and continuous replenishment from the thymus.