IL-18 is produced during the acute immune response by macrophages and immature dendritic cells. IL-18 receptors are induced on T cells and NK cells by IL-12 and together they enhance a cellular immune response. We constructed retroviral and adenoviral vectors encoding the mature bioactive murine IL-18 in order to examine their immune and antitumor effects in murine tumor models. Secretion of bioactive IL-18 from murine tumor cells was facilitated by transfecting them with recombinant viral vectors carrying the prepro leader sequence of human parathyroid hormone fused to the 5' end of the mature murine IL-18 cDNA. Direct injection of the IL-18 recombinant adenoviral vector (Ad.PTH.IL-18) into an established MCA205 murine fibrosarcoma completely eradicated tumor in all animals with concomitant induction of protective systemic immunity. Co-administration of systemic IL-12 provided synergistic antitumor effects when combined with peritumoral injections of Ad.PTH.IL-18 without apparent side-effects as we observed with systemic administration of IL-18. Depletion of asialo GM-1+ cells completely abrogated the antitumor effects of Ad.PTH.IL-18, suggesting a major role for NK cells in mediating the anti-tumor effects of IL-18. Peritumoral injection of Ad.PTH.IL-18 was also associated with reduced numbers of CD8+ cells found within the tumor (HBSS versus Ad.PTH.IL-18, P < 0.0001). This suggests that IL-18 could be utilized as an alternative cancer gene therapy especially when combined with systemic administration of IL-12.