Driving high-level transgene expression in a tumour-specific manner remains a key requirement in the development of cancer gene therapy. We have previously demonstrated the strong anticancer effects of generating abnormally high levels of intracellular NO center dot following the overexpression of the inducible nitric oxide synthase (iNOS) gene. Much of this work has focused on utilizing exogenously activated promoters, which have been primarily induced using X-ray radiation. Here we further examine the potential of the pE9 promoter, comprising a combination of nine CArG radio-responsive elements, to drive the iNOS transgene. Effects of X-ray irradiation on promoter activity were compared in vitro under normoxic conditions and various degrees of hypoxia. The pE9 promoter generated high-level transgene expression, comparable with that achieved using the constitutively driven cytomegalovirus promoter. Furthermore, the radio-resistance of radiation-induced fibrosarcoma-1 (RIF-1) mouse sarcoma cells exposed to 0.1 and 0.01% O-2 was effectively eliminated following transfection with the pE9/iNOS construct. Significant inhibition of tumour growth was also observed in vivo following direct intratumoural injection of the pE9/iNOS construct compared to empty vector alone (P < 0.001) or to a single radiation dose of 10 Gy (P < 0.01). The combination of both therapies resulted in a significant 4.25 day growth delay compared to the gene therapy treatment alone (P < 0.001). In summary, we have demonstrated the potential of the pE9/ iNOS construct for reducing radio-resistance conferred by tumour cell hypoxia in vitro and in vivo, with greater tumour growth delay observed following the treatment with the gene therapy construct as compared with radiotherapy alone.