Single-fraction radiation therapy with 5 or 15 Gy ⁶⁰Co γ radiation was combined with intraperitoneal injections of syngeneic interferon gamma (IFN-γ)-transfected cells in rats with intracerebral N29 or N32 glioma tumors at days 7, 21 and 35 after inoculation. For intracerebral N29 tumors, single-fraction radiation therapy with 5 or 15 Gy had no significant effect on the survival time. Immunization with IFN-γ-transfected N29 cells significantly increased the survival time by 61%. Single-fraction radiation therapy with 5 Gy combined with immunization increased the survival time significantly by 87% and complete remissions by 75% while with 15 Gy the survival time increased 45% with 38% complete remissions. For intracerebral N32 tumors, single-fraction radiation therapy with 15 Gy increased the survival time significantly by 20%. Immunization by itself had no significant effect with IFN-γ-transfected N32 cells, but combined with 15 Gy single-fraction radiation therapy it increased survival time significantly by 40%, although there were no complete remissions. Based on these findings, we suggest a new therapeutic regimen for malignant glioma using single-fraction radiation therapy with a target absorbed dose of the order of 5–10 Gy combined with clinically verified immunotherapy.