The radiosensitizing effect of inducible nitric oxide synthase (iNOS) was evaluated, in vitro , in a feline vaccine-associated sarcoma (VAS) cell line and a canine osteosarcoma cell line (D17). The gene encoding the human iNOS was cloned into an expression plasmid under the control of a cytomegalovirus immediate early promoter. Transient transfections were performed in feline VAS cells and D17 cells. Nitric oxide was measured in the supernatant media 48h later as an indirect measurement of iNOS expression. Cells were irradiated using cobalt-60 under hypoxic or oxic conditions, and clonogenic assays were used to evaluate the effects of gene transfer on the sensitivity of cells to radiation. The results demonstrated that iNOS had no significant effect on improving the radiosensitivity of cells under oxic conditions. However, under hypoxic conditions, iNOS gene transfer significantly improved radiosensitization in osteosarcoma cells. These results demonstrate the feasibility of improving the outcome of radiotherapy in dogs with large bulky tumours using iNOS gene therapy.