Dividing cells and non-dividing cells are distinct in their cell cycle kinetics, and react differently when facing cytotoxic stimuli. A protein synthesis inhibitor, cycloheximide (CHX), has recently been found to protect neuronal cells from oxidative stress. We investigated whether CHX exerts differential effects on dividing and non-dividing cells in the brain under cytotoxic stimuli. Mitotic C6 rat glioma cells and postmitotic neuronal cells were studied with a cytotoxic regimen combining gamma-irradiation (RT) and 1,3-bis,2-chloroethyl-1-nitrosourea (BCNU). Cells were exposed to BCNU (1 g/ml) for 15 h before gamma-irradiation and incubated with CHX (1 g/ml) from 30 min before and until 5 h after irradiation. Clonogenic assay was used to assess cytotoxic effects on C6 glioma cells. LDH assay was used for the viability of H19-7 postmitotic neuronal cells. A 2.27-3.75 fold enhancement of cytotoxicity was noticed with the addition of CHX to BCNU and 2-10 Gy of radiation. Our data demonstrated that CHX enhanced cytotoxicity of RT plus BCNU, while no additional toxicity was incurred to the postmitotic neuronal cells when CHX was added. We further studied whether the inhibition of DNA repair, assayed by single-cell DNA electrophoresis (comet assay), is a contributing factor for the enhanced cytotoxicity on C6 glioma cells. Interestingly, the initial DNA damage after RT plus BCNU was equivalent; whereas DNA repair was significantly less at 5 h after radiation in CHX-treated C6 glioma cells. Protecting non-dividing neuronal cells to avoid excessive functional deficit is an integral part of a successful brain tumor treatment regimen. Taking advantage of the differential effect of CHX on glioma and neuronal cells may improve tumor control without excessive neural toxicity.