Recent studies have emphasized the importance of T cells with regulatory/suppressor properties in controlling autoimmune diseases. A number of different types of regulatory T cells have been described with the best characterized being the CD25+ population. In addition, it has been shown that regulatory T cells can be induced by specific Ag administration. In this study, we investigate the relationship between peptide-induced, CD4+ regulatory T cells and naturally occurring CD4+ CD25+ cells derived from the Tg4 TCR-transgenic mouse. Peptide-induced cells were FoxP3− and responded to Ag by secreting IL-10, whereas CD25+ cells failed to secrete this cytokine. Both cell types were able to suppress the proliferation of naive lymphocytes in vitro although with distinct activation sensitivities. Depletion of CD25+ cells did not affect the suppressive properties of peptide-induced regulators. Furthermore, peptide-induced regulatory/suppressor T cells could be generated in RAG−/−, TCR-transgenic mice that do not spontaneously generate CD25+ regulatory cells. These results demonstrate that these natural and induced regulatory cells fall into distinct subsets.