Although murine tumor cells have been transfected to express a multitude of different cytokines and shown to be rejected in vivo, it is unclear which of these factors might be useful to facilitate tumor Ag immunization schemes. A study of the normal immune mechanisms involved in tumor rejection when it naturally occurs should reveal critical signals for generation of antitumor CTL in vivo. The highly transfectable variant of P815, P1.HTR, was found to be rejected in the hind footpads by approximately one-third of syngeneic DBA/2 mice. Analysis of draining popliteal lymph nodes revealed a large influx of CD4+ and CD8+ T lymphocytes in all mice, indicating that a failure to reject was not due to the complete absence of an inflammatory response. However, although IL-2 and IL-3 were produced by lymph node cells from all mice, only approximately one-third generated a high IFN-gamma response. IL-4 was not detected. To explore a role for IL-12 in the induction of the IFN-gamma-producing phenotype, a histidine-tagged IL-12 fusion protein was expressed in mammalian cells and purified by nickel-chelate chromatography, and a rabbit antiserum was produced. Neutralization of IL-12 in vivo eliminated the high IFN-gamma response and prevented rejection of P1.HTR tumors and also of a more immunogenic tum- variant of P815, P198. Conversely, exogenous IL-12 delivered early during challenge with P1.HTR cells induced high IFN-gamma production and resulted in tumor rejection in most mice. Therefore, endogenous IL-12 is vital for the rejection of these tumors when it naturally occurs, supporting a role for exogenous administration of this cytokine to favor a Th1-like phenotype in the immunotherapy of cancer.