We have previously demonstrated that carbamazepine (CBZ) at concentrations above the therapeutic range is toxic to cultured cerebellar granule cells. Here, we ask whether the effect of CBZ involves neuronal apoptosis or necrosis. Treatment of cultured cerebellar granule cells with CBZ for 3 days resulted in a concentration-dependent fragmentation of DNA revealed as a laddered pattern in agarose gel electrophoresis, a phenomenon characteristic of apoptosis. Pretreatment of cells with N-methyl-D-aspartate (NMDA) blocked CBZ-induced DNA fragmentation and neurotoxicity as assayed by loss of mitochondrial activity with MTT or by [3H]ouabain binding to Na+/K(+)-ATPase. Aurintricarboxylic acid (ATA), a polyanionic dye, also markedly suppressed DNA fragmentation and cell death detected by morphological examination. A considerable level of DNA ladder formation was detected in untreated cells and this basal DNA fragmentation was also blocked by NMDA and ATA. Moreover, NMDA and ATA prevented CBZ-induced chromatin condensation as revealed by DNA binding with the fluorescent dye Hoechst 33258. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, prevented CBZ-induced cell death detected morphologically and attenuated CBZ-induced neurotoxicity assessed by mitochondrial activity and [3H]ouabain binding assays. Taken together, our results suggest that CBZ-induces death of cerebellar granule cells by an apoptotic process that is sensitive to NMDA, ATA and cycloheximide.