The functions of NK cells are regulated by the balance of activating and inhibitory signals. The inhibitory NK cell receptors are well understood; however, less is known about the activating signaling pathways. To explore whether a costimulatory receptor, inducible costimulator (ICOS), is involved in NK cell function, we assessed the role of ICOS in NK cell-mediated cytotoxicity and cytokine production. In addition, to determine whether ICOS contributes to the elimination of tumors in vivo, we examined the tumor growth survival of mice injected with a tumor expressing the ICOS ligand, B7RP-1. We found that ICOS was up-regulated by cytokine stimulation in murine NK cells. Consistent with ICOS expression on activated NK cells, ICOS-dependent cytotoxicity and IFN-gamma production were observed, and appeared to require signaling through the phosphoinositide 3-kinase pathway. Interestingly, ICOS-mediated stimulation allowed activated NK cells to kill more efficiently tumor cells expressing MHC class I. Furthermore, fewer metastases appeared in the liver and spleen of mice injected with the ICOS ligand-expressing tumor compared with mice bearing the parental tumor. These results indicate that NK cell functions are regulated by ICOS.