Regulatory T cells (Tregs) play an important role in the maintenance of immune tolerance to self-antigens and are involved in modulating immune responses in autoimmunity, transplant rejection, and tumor immunity. Recently, a novel subset of TCR-αβ(+) CD4(-) CD8(-) (double negative, DN) T cells has been described to specifically suppress T-cell responses in mice. Here, we demonstrate that human DN T cells are highly potent suppressors of both CD4(+) and CD8(+) T-cell responses. In contrast to naturally occurring CD4(+) CD25(+) Tregs, DN T cells have to be activated by antigen-presenting cells (APCs) to induce their regulatory potential. The suppressive activity of DN T cells is neither mediated indirectly by modulation of APCs nor by competition for T-cell growth factors. Furthermore, DN T-cell-mediated suppression toward responder T cells is TCR dependent and requires novel protein synthesis. In contrast to murine DN T cells, which eliminate effector T cells via Fas/FasL or perforin/granzyme, human DN T cells suppress proliferation of responder T cells by cell contact-dependent mechanisms. Taken together, our data indicate that human DN T cells exert strong immunosuppressive effects on both CD4(+) and CD8(+) T cells and may serve as a new therapeutic approach to treat autoimmunity and transplant rejection.