One of the most promising approaches for cancer gene therapy is the use of the so-called suicide genes, which encode prodrug-activating enzymes and render transduced cells more sensitive to prodrugs. The enzyme nitroreductase (NTR) converts prodrug CB1954 into a cytotoxic DNA interstrand cross-linking agent. We have established transgenic mice in which the pro-oncogene c-myc and NTR were fused to the internal ribosome entry site and coexpressed in luminal cells of the mammary gland under the control of mouse whey acidic protein (WAP) promoter to evaluate NTR mediated ablation of mammary tumors. More than 78% of transgenic females developed in situ or infiltrating carcinomas after three to four pregnancies. By contrast, if the transgenic female mice were given the prodrug CB1954 during their third lactation, the incidence of tumors decreased to less than 40% (P < 0.05). The total number of carcinomas was even more striking with 117 carcinomas identified in 14 non-ablated transgenics compared with only five in 15 treated animals (p < 0.05, student t test). C-myc induced pleomorphic nuclei and mitotic figures were seen as a field change in over 70% of the untreated transgenics compared to 20% in the treated group. Our results suggest that the enzyme pro-drug system NTR-CB1954 efficiently inhibit myc-dependent tumor formation and malignant progression in the mammary gland.