The DNA Comet assay (single gel electrophoresis) is a rapid, inexpensive, simple and sensitive technique for measuring and analysing DNA breakage in individual cells. This technique has many applications in radiation biology in that the physicochemical interaction of radiation and cellular DNA produces a variety of primary lesions such as single- and double- DNA strand breaks.   Radiation therapy is one of the effective choices available for cancer treatment. Since tumour tissues have different radiosensitivities, it is important to determine the intrinsic radiosensitivity of tumours before radiotherapy. In order to validate Comet assay for detection of DNA damage caused by radiotherapy, we exposed murine glioblastoma cells (RT2) to gamma radiation (⁶⁰Co) in vitro. RT2 cells treated with gamma radiation (9Gy) or control were submitted to DNA analysis. Comet assay was done in alkaline conditions and stained by silver staining method. The extent of DNA damage could be assessed from the length of DNA migration towards the anode, which was derived by subtracting the diameter of the nucleus from the total length of the image. The results were compared with data obtained by DNA Ladder assay. The treatment with 9Gy of gamma radiation evoked DNA damage in RT2 cells immediately after the irradiation. A significant increase in the mean number of cells with tailed DNA migration was found between the control and exposed groups; moreover, the length of the tails observed in treated cells was higher than that observed in control cells (P< 0.01). Our results show that DNA Comet assay was as sensitive as DNA Ladder assay for detection of DNA damage produced immediately after treatment with gamma radiation in RT2 cells. Furthermore, Comet assay is more inexpensive and simpler. DNA Comet assay proved to be useful for detect genotoxic effect of gamma radiation in murine glioblastoma cells. Further studies are in development to verify the correlation between DNA damage progression and time after radiotherapy.