The radiosensitizing effect of nitric oxide (NO) on mouse and human tumor cells with different degrees of malignancy was evaluated. Cells pre-treated with the NO donor diethylenetriamine-NO (DETA-NO), were irradiated with gamma rays. Survival curves were obtained by clonogenicity and fitted to the linear-quadratic model. Results demonstrated an association between radiosensitization and degree of malignancy. The more malignant the cell line, the higher the degree of radiosensitization by DETA-NO. In conclusion, the differential radiosensitizing effect of DETA-NO shown here is of great interest for the potential use of NO in radiotherapy, due to an enhanced radiation effect on tumor vs. normal tissue.