Background Inducible nitric oxide synthase (iNOS) gene therapy has been identified as a potential anti-tumour strategy. A major problem common to most gene therapy strategies is targeting of treatment to the turnout volume. in this study we report on the use of the X-ray-inducible WAF1 promoter to achieve targeting of iNOS expression to the turnout volume. Methods A WAF1/iNOS/liposome complex was injected directly into RIF-1 and HT29 tumours in mice. A 4 Gy dose of X-rays was applied to induce the WAF1 promoter followed, 8 h later, by treatment doses of 10 or 20 Gy. Tumour volume was measured, and growth curves plotted. Results Intra-tumoural injection of WAF1/iNOS combined with a priming dose of X-rays to induce the WAF1 promoter, followed by a treatment dose, resulted in sensitiser enhancement ratios of 2.0 and 1.3 in RIF-1 and HT29 tumours, respectively, compared with radiation treatment alone. PCR analysis of organ tissue after intra-tumoural injection of WAF1/iNOS showed that vector sequences were detected in all tissue tested; however, Western blot analysis revealed that iNOS protein levels were significantly increased only in tumour and the surrounding dermal tissue that had been exposed to the 4 Gy inducing dose. Conclusions iNOS gene therapy in combination with an inducible promoter results in significant tumour cell radiosensitisation. The WAF1 promoter may be a good candidate for a gene therapy as it is silent in normal tissue yet can be induced by the tumour environment. Copyright (C) 2004 John Wiley Sons, Ltd.