Amongst the many types of regulatory cells that have been described during the past few years, the spontaneously occurring population that is characterized by co-expression of CD4 and CD25 appears to play a key role in the prevention of autoimmunity and the maintenance of transplantation tolerance. In this study we have examined the ability of CD4+CD25+ T cells to regulate human CD8+ T cells, and the behavior of CD8+ T cells following activation in the presence of regulatory CD4+CD25+ T cells. The experiments described here demonstrate that human CD4+CD25+ T cells cause pronounced and sustained inhibition of CD8+ T cell proliferation in response to polyclonal and allogeneic stimulation. The regulation of CD8+ T cell activation was cell contact-dependent and included inhibition of perforin, granzyme B and IFN-γ cytokine production at the transcriptional level and impaired cytotoxicity. The regulated CD8+ T cell population showed sustained hyporesponsiveness and refractoriness to exogenous IL-2. These data provide insights into the short- and long-term effects of CD4+CD25+ T cells on CD8+ T cells that could be of considerable value in optimizing vaccination against tumor and viral antigens.