CD4+ CD25+ regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4+ CD25+ regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4+ CD25+ regulatory T cells anergize CD4+ T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4+ CD25+ T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4+ and CD8+ T cells. This suppression only occurs when CD4+ CD25+ T cells are preactivated. Furthermore, we could demonstrate that CD4+ T-cell clones stop secreting interferon-γ (IFN-γ), start to produce interleukin-10 and transforming growth factor-β after coculture with preactivated CD4+ CD25+ T cells and become suppressive themselves. Surprisingly preactivated CD4+ CD25+ T cells affect CD8+ T cells differently, leading to reduced proliferation and reduced production of IFN-γ. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8+ T cells, unlike CD4+ T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4+ CD25+ T cells.