To induce cytolytic immunity, dendritic cells (DCs) need to release bioactive interleukin-12 (IL-12) p70 heterodimeric molecules. To study the role of IL-12 for the generation of an anti-tumor immune response, we generated two classes of DCs. (1) DCs were initiated to secrete IL-12 by exposure to LPS/IFN-gamma for 2 h resulting, as demonstrated in vitro, in continued IL-12 release for another 24 h (termed active DCs). (2) DCs were exposed to LPS/IFN-gamma for 24 h and injected into mice at a time point when IL-12 production had ceased (termed exhausted DCs). These two classes of DCs were probed for their capacity to induce a cytolytic anti-tumor immune response in vivo in a syngeneic mouse tumor model. The mouse tumor cell line K-Balb was engineered to express neomycin phosphotransferase (NPT) as a model tumor antigen. DCs were charged with various NPT-derived antigens, including recombinant NPT protein, whole tumor cell lysate and NPT-derived synthetic peptides, and the induction of in vivo anti-tumor immunity was determined by measuring tumor growth. Only the injection of active DCs, i.e., cells that maintained the capacity to secrete IL-12, but not exhausted DCs that had lost the ability to produce IL-12, resulted in a measurable deceleration of growth of K-Balb-NPT tumors. This anti-tumor immune response was most pronounced when using recombinant protein as an antigen source, which was evident in a prophylactic as well as in a therapeutic setting. The absence of a response to parental K-Balb tumors confirmed the antigen specificity of the anti-tumor immune response. Together these data provide evidence for the unique capacity of actively IL-12 secreting DCs to trigger effective anti-tumor immunity using exogenous tumor antigens.