Despite the use of multimodal treatments incorporating surgery, chemotherapy and radiotherapy, local control of gliomas remains a major challenge. The potential of a new treatment approach called indirect radio-chemo-beta therapy using the synergy created by combining methotrexate (MTX) with bromodeoxyuridine (BrUdR) under optimum energy x-ray irradiation is assessed. 9L rat gliosarcoma cells pre-treated with 0.01 μM MTX and/or 10 μM BrUdR were irradiated in vitro with 50 kVp, 125 kVp, 250 kVp, 6 MV and 10 MV x-rays. The cytotoxicity was assessed using clonogenic survival as the radiobiological endpoint. The photon energy with maximum effect was determined using radiation sensitization enhancement factors at 10% clonogenic survival (SER10%). The cell cycle distribution was investigated using flow cytometric analysis with propidium iodide staining. Incorporation of BrUdR in the DNA was detected by the fluorescence of labelled anti-BrUdR antibodies. The radiation sensitization enhancement exhibits energy dependence with a maximum of 2.3 at 125 kVp for the combined drug treated cells. At this energy, the shape of the clonogenic survival curve of the pharmacological agents treated cells changes substantially. This change is interpreted as an increased lethality of the local radiation environment and is attributed to supplemented inhibition of DNA repair. Radiation induced chemo-beta therapy was demonstrated in vitro by the targeted activation of combined pharmacological agents with optimized energy tuning of x-ray beams on 9 L cells. Our results show that this is a highly effective form of chemo-radiation therapy.