The efficacy of radiotherapy for cancer is limited by the dose that can be safely delivered to the tumor without causing debilitating side effects. Previous studies have shown an additive or syngeneic reduction in the volume of malignant tumors when tumor necrosis factor-alpha (TNF-alpha) protein is administered prior to radiation. The major goal of the present investigation was to evaluate the efficacy of pGL1-TNF-alpha, a new plasmid construct that expresses human TNF-alpha protein, together with radiotherapy in the C6 glioma/athymic mouse model. Subcutaneously growing tumors were injected with pGL1-TNF-alpha complexed with a cationic polyamine and radiation, singly and in combination, over an 8-day period. The maximum antitumor effect was achieved with the combination of polyamine-pGL1-TNF-alpha and radiation. Each modality used alone, including polyamine, modestly slowed tumor growth. In vitro evaluations of blood, spleen, and tumor indicated that the antitumor mechanisms of combination therapy may include, at least partly, the recruitment and activation of nonspecific effector cells. The results demonstrate that polyamine-pGL1-TNF-alpha can be safely and effectively administered together with radiation under the conditions used.