Dendritic cells (DCs) are potent antigen-presenting cells that can prime and boost systemic antitumor immunity. Here, we have evaluated the ability of DCs transfected to secrete the potent Th1-biasing cytokine interleukin (IL)-18 to promote enhanced antitumor immunity in a mouse sarcoma model. DCs infected with a recombinant adenovirus encoding IL-18 (AdIL18DC) expressed higher levels of MHC and costimulatory molecules and were better stimulators than control DCs in mixed leukocyte reactions in vitro. Immunization of BALB/c mice bearing established day 7 CMS4 tumors with tumor peptide-pulsed control Adpsi5-transfected DCs or nontransduced DCs significantly inhibited the growth of established tumors but did not lead to complete regression of established tumors. Importantly, immunization with antigen-loaded AdIL18DC resulted in tumor rejection or further suppression of tumor growth when compared with controls. The repertoire of naturally presented tumor peptides recognized by splenocytes (as deduced in IFN-gamma ELISA assays) from AdIL18DC-treated animals was far more diverse and of greater magnitude than that of all other groups, in association with improved therapeutic outcome. These results support the ability of IL-18 gene transfer to enhance the capacity of DCs to drive broadly reactive Th1-type therapeutic immunity prompted by single peptide epitope-based vaccines (i.e., epitope spreading).