CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. These cells have been reported to be capable of suppressing the response of CD4+CD25- T cells in vitro. The depletion of these cells evokes effective immune responses to tumor cells in vivo. In this study, we demonstrate that CD4+CD25+ T cells also suppress all subsets of Valpha24+NKT cells (Valpha24+CD4-CD8- double negative, Valpha24+CD4+, and Valpha24+CD8+) in both proliferation and cytokine production [IFN-gamma, interleukin-4 (IL-4), IL-13, and IL-10]. This suppression is mediated by cell-to-cell contact but not by a humoral factor or the inhibition of antigen-presenting cells. Moreover, the cytotoxic activity of Valpha24+NKT cells against some tumor cell lines is suppressed by CD4+CD25+ T cells. This finding is important in developing an effective immunotherapy for cancer.