CD4+ regulatory T cells (Tregs) accumulate at tumor sites and play a critical role in the suppression of immune responses against tumor cells. In this study, we show that two immunodominant epitopes derived from the tumor Ags (TAs) NY-ESO-1 and TRAG-3 stimulate both CD4+ Th cells and Tregs. TA-specific Tregs inhibit the proliferation of allogenic T cells, act in a cell-to-cell contact dependent fashion and require activation to suppress IL-2 secretion by T cells. TRAG-3 and NY-ESO-1–specific Tregs exhibit either a Th1-, a Th2-, or a Th0-type cytokine profile and dot not produce IL-10 or TGF-β. The Foxp3 levels vary from one Treg clone to another and are significantly lower than those of CD4+ CD25 high Tregs. In contrast to NY-ESO-1–specific Th cells, the NY-ESO-1–specific and TRAG-3–specific Treg clonotypes share a common TCR CDR3 Vβ usage with Foxp3+ CD4+ CD25 high and CD4+ CD25− T cells and were not detectable in PBLs of other melanoma patients and of healthy donors, suggesting that their recruitment occurs through the peripheral conversion of CD4+ CD25− T cells upon chronic Ag exposure. Collectively, our findings demonstrate that the same epitopes spontaneously stimulate both Th cells and Tregs in patients with advanced melanoma. They also suggest that TA-specific Treg expansion may be better impaired by therapies aimed at depleting CD4+ CD25 high Tregs and preventing the peripheral conversion of CD4+ CD25− T cells.