Evidence suggests that regulatory T cells expressing the transcription factor Foxp3 develop extrathymically and intrathymically. Mechanisms of extrathymic induction require further scrutiny, especially as proliferation and/or phenotypic changes of preexisting suppressor cells must be distinguished from true de novo generation. Here we report the conversion of truly naive CD4(+) T cells into suppressor cells expressing Foxp3 by targeting of peptide-agonist ligands to dendritic cells and by analysis of Foxp3 expression at the level of single cells. We show that conversion was achieved by minute antigen doses with suboptimal dendritic cell activation. The addition of transforming growth factor-beta or the absence of interleukin 2 production, which reduces proliferation, enhanced the conversion rate. In addition, regulatory T cell populations induced in subimmunogenic conditions could subsequently be expanded by delivery of antigen in immunogenic conditions. The extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immunity.