Normal T cell repertoire contains regulatory T cells that control autoimmune responses in the periphery. One recent study demonstrated that CD4+ CD25+ T cells were generated from autoreactive T cells without negative selection. However, it is unclear whether, in general, positive selection and negative selection of autoreactive T cells are mutually exclusive processes in the thymus. To investigate the ontogeny of CD4+ CD25+ regulatory T cells, neo-autoantigen-bearing transgenic mice expressing chicken egg OVA systemically in the nuclei (Ld-nOVA) were crossed with transgenic mice expressing an OVA-specific TCR (DO11. 10). Ld-nOVA× DO11. 10 mice had increased numbers of CD4+ CD25+ regulatory T cells in the thymus and the periphery despite clonal deletion. In Ld-nOVA× DO11. 10 mice, T cells expressing endogenous TCR αβ chains were CD4+ CD25− T cells, whereas T cells expressing autoreactive TCR were selected as CD4+ CD25+ T cells, which were exclusively dominant in recombination-activating gene 2-deficient Ld-nOVA× DO11. 10 mice. In contrast, in DO11. 10 mice, CD4+ CD25+ T cells expressed endogenous TCR αβ chains, which disappeared in recombination-activating gene 2-deficient DO11. 10 mice. These results indicate that part of autoreactive T cells that have a high affinity TCR enough to cause clonal deletion could be positively selected as CD4+ CD25+ T cells in the thymus. Furthermore, it is suggested that endogenous TCR gene rearrangement might critically contribute to the generation of CD4+ CD25+ T cells from nonautoreactive T cell repertoire, at least under the limited conditions such as TCR-transgenic models, as well as the generation of CD4+ CD25− T cells from autoreactive T cell repertoire.