5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy and fluorescence-guided resection of malignant gliomas due to its high cellular uptake in tumours. Porphyrin compounds act not only as photosensitizers but also as radiosensitizers. In the present study, the possible use of 5-ALA as a radiosensitizer for malignant gliomas was examined in vitro. Rat glioma cell lines (9L, C6) were pre-treated with 5-ALA and exposed to ionizing irradiation. The radiosensitizing effect of 5-ALA was evaluated by colony-forming assay. Intracellular reactive oxygen species (ROS) produced by 5-ALA and irradiation were evaluated by confocal laser scanning microscopy. Pre-treatment with 5-ALA enhanced the radiosensitivity of 9L cells to single-dose ionizing irradiation compared with controls (D0 value, 4.35 ± 0.20 and 4.84 ± 0.23 Gy, respectively, P ≤ 0.05). Exposure to multi-dose ionizing irradiation revealed high radiosensitivity in both 9L and C6 cells pre-treated with 5-ALA compared to controls. Production of intracellular ROS increased in 9L cells pre-treated with 5-ALA after ionizing irradiation compared to control cells. Thus, 5-ALA functions as a specific radiosensitizer for malignant gliomas. Intracellular 5-ALA-induced PpIX plays an important role in the production of ROS and the radiosensitizing effect under ionizing irradiation conditions.