Radiosurgery is used increasingly upon recurrence of high-grade gliomas to deliver a high dose of focused radiation to a defined target. The purpose of our study was to compare intermittent irradiation (IIR) by using a CyberKnife (CK) with continuous irradiation (CIR) by using a conventional linear accelerator (LINAC). A significant decrease in surviving fraction was observed after IIR irradiation compared with after CIR at a dose of 8 Gy. Three hours after irradiation, most of the DNA damage was repaired in U87. Slightly higher basal levels of Ku70/80 mRNA were found in U87 compared with A172, while radiation treatment induced only minor regulation of Ku70/80 and Rad51 transcription in either cell lines. IIR treatment using CK significantly decreased the survival in U87 and A172 compared with CIR. Although the two cell lines differed in DNA repair capability, the role of Ku70/80 and Rad51 in the cell line radiosensitivity seemed marginal.