Cancer gene therapy that utilizes toxic transgene products requires strict transcriptional targeting to prevent adverse normal tissue effects. We report on the use of a promoter derived from the cyclin dependent kinase inhibitor, p21((WAF1)), to control transgene expression. We demonstrate that this promoter is relatively silent in normal cells (L132, FSK, HMEC-1) compared to the almost constitutive expression obtained in tumour cells (DU145, LNCaP, HT29 and MCF-7) of varying p53 status, a characteristic that will be important in gene therapy protocols. In addition, we found that the p21(WAF1) promoter could be further induced by both external beam radiation (up to eight-fold in DU145 cells), intracellular-concentrated radionuclides ([At-211] MABG) (up to 3.5-fold in SK-N-BE(2c) cells) and hypoxia (up to four-fold in DU145 cells). We have previously achieved significant radiosensitization of tumour cells both in vitro and in vivo by using inducible nitric oxide synthase (iNOS) gene therapy to generate the potent radiosensitizer, nitric oxide (NO center dot). Here, we report that a clinically relevant schedule of p21((WAF1))-driven iNOS gene therapy significantly sensitized both p53 wild-type RIF-1 tumours and p53 mutant HT29 tumours to fractionated radiotherapy. Our data highlight the utility of this p21((WAF1))/iNOS-targeted approach.