Free radical (FR) production, a major step in calcium-dependent neurodegeneration, has been linked to the generation of epileptiform activity and seizure-induced cell death. However, direct evidence of FR production in neurons during seizures has never been presented. Using hippocampal cultured slices we demonstrate that FRs are produced in CA3 but not CA1 pyramidal neurons during the rhythmic synchronous activity induced by the GABAA receptor antagonist bicuculline. The production of FRs (measured as changes in the fluorescence emission of dihydrorhodamine 123) was correlated with an increase in the baseline levels of intracellular calcium ([Ca2+]i) estimated by fluo-3 injected into individual neurons via a patch pipette. [Ca2+]i increased during spike bursting and returned to baseline levels after the burst termination in CA1, but not in CA3, pyramidal neurons where ‘interburst’ calcium concentrations progressively increased. Measurement of cell death, performed with propidium iodide 48 h after a 30-min exposure to bicuculline, revealed most prominent degeneration of pyramidal neurons in the CA3 pyramidal layer. The FR scavengers vitamin E and glutathione significantly reduced the seizure-induced neurodegeneration without supressing spontaneous epileptiform activity. These observations indicate that FR overproduction is related to seizure-induced neuronal death.