OBJECTIVE: Interleukin-12 (IL-12) may be useful for immunotherapy against gliomas because it can reverse the glioma-induced suppression of T- cell proliferation and interferon-γ production. We postulated that peripheral infusion of IL-12 along with irradiated tumor cells can lead to immunological rejection of 9L glioma. METHODS: 9L gliosarcoma flank tumors were established in syngeneic Fischer 344 rats. Osmotic minipumps delivered IL-12 subcutaneously, and irradiated 9L cells were injected on Days 0, 3, 7, 14, and 21. Tumor volumes were measured by a blinded observer. For tumor rechallenge, animals initially cured of 91 flank tumors received either another implantation of flank tumor or a stereotactic injection of 106 9L cells into the right striatum. Delayed-type hypersensitivity was measured after injecting 106 irradiated 9L tumor cells into the right pinnae. RESULTS: Tumor growth curves were significantly different between treated and control animals. Among the animals that received 1 ng per day of IL-12, 40% did not develop any measurable tumors at all. A combination of irradiated 9L cells and IL-12 was necessary for optimal effect. Cured animals rejected future flank tumors. All animals rechallenged with intraparenchymal brain tumors survived, whereas control animals all died by Day 22. Delayed-type hypersensitivity measurements showed a specific and long-lasting response against 9L cells. CONCLUSION: Continuous administration of the lymphokine IL- 12, in the presence of irradiated tumor cells for antigen presentation, circumvents the need for gene transfection for generating tumor cell vaccines. We have demonstrated that the combination of IL-12 and irradiated tumor cells can lead to regression of 9L flank tumors and resistance to future flank and central nervous system tumor challenges.