Immunoregulatory CD25+CD4 T cells are thought to arise from the thymus as a distinct lineage of CD4 T cells specific for self Ags. We used the DO11.10 TCR transgenic adoptive transfer system to show that cells of similar phenotype may also arise in the course of peripheral tolerance induction. Such cells emerged within 1 wk following Ag exposure and correlated negatively with the number of initial cell divisions. Limiting i.v. Ag dose or using an oral tolerance protocol yielded the greatest numbers of Ag-specific CD25+CD4 T cells. In contrast, immunogenic Ag exposure in the presence of an adjuvant did not lead to emergence of CD25+CD4 T cells. The profound anergic phenotype of these cells and their potential immunoregulatory properties make them an especially desirable population to induce in the course of immunotherapy in numerous clinical settings. This experimental system may be useful in future studies designed to optimize immunologic tolerance induction.