The fact that antioxidants reduce some types of brain damage mediated by glutamate receptors suggests that reactive oxygen species (ROS) may have a role. In order to determine whether ROS could increase in the event of glutamate neurotoxicity in rat organotypic hippocampal cultures, we observed (1) morphological changes of the neurons in the slice following glutamate (1 mM) exposure under a video-enhanced microscope and (2) production of ROS elicited by glutamate (1 mM) in the cultured slice. Lucigenin is well recognized for its ability to react with ROS, yielding a product that emits chemiluminescence (CL). By measuring lucigenin (300 μM)-dependent CL with a photomultiplier, the production of ROS was continuously monitored in the cultured slice. Under a high-resolution microscope we observed rapid nuclear changes of the neurons 10 to 20 min after glutamate exposure. However, ROS did not increase but decrease during the same period following exposure to glutamate.