The mammalian RAD51 gene is a homologue of the yeast RAD51 and E. coli RecA genes, which are related to the repair of DNA double-strand breaks and are also involved in recombination repair and various SOS responses to DNA damage by gamma-irradiation and alkylating reagents. In this study, we investigated both in vitro and in vivo whether inhibition of the RAD51 gene by antisense oligonucleotides (ODNs) enhances the radiosensitivity of mouse malignant gliomas. A volume of 100 nM of RAD51 antisense ODNs inhibited the level of mRNA by more than 95% and reduced the protein expression by about 70%. Treatment of mouse 203G glioma cells with 100 nM of RAD51 antisense ODNs significantly enhanced the radiation-induced cell kill compared to control cells, and cells treated with sense or scrambled ODNs. When the glioma cells were implanted in the cisterna magna of mice followed by treatment with RAD51 antisense ODNs, the survival time of the mice was markedly prolonged compared to that of the untreated group (p < 0.001, logrank test). In addition, the combination of antisense ODNs and irradiation extended the survival time of the glioma-bearing mice much longer than could be achieved with radiation alone (p < 0.0001, logrank test). These results suggest that inhibition of RAD51 can be expected to serve as a novel potentiator for radiation therapy in malignant gliomas by inhibiting DNA double-strand break repair.