CD(4) (+)CD(25) (+) regulatory T cells (Tregs) have been shown to inhibit cytotoxic lymphocytes-mediated immune responses. Cytokine-induced killer (CIK) cells exert high impact on adoptive immunotherapeutic approaches. Therefore, the purpose of this report was to determine the effect of Tregs on CIK cell growth and CIK-induced cytotoxicity for inhibition of tumor growth in vivo as well as in vitro. After depletion of CD(4) (+)CD(25) (+) cells before culture, the proliferation and cytotoxicity of CIK cells, which indicated in bromodeoxyuridine (BrdU) and lactic dehydrogenase (LDH) assays, were significantly increased. Depletion of CD(4) (+)CD(25) (+) cells preculture also enhanced the suppression effect on the lung cancer cells inoculated in experimental animals. Blockage of glucocorticoid-induced tumor necrosis factor receptor (GITR) and transforming growth factor beta1 (TGF-beta1) by antibodies partially abrogated the suppressive effect of CD(4) (+)CD(25) (+) cells on CIK. These results indicated that Tregs could inhibit the antitumor activity of CIK cells. The molecules TGF-beta and GITR may contribute to the suppressive function of CD(4) (+)CD(25) (+) cells.