The aim of this study was to elucidate the mechanism underlying the neurotoxic effect of beta-bungarotoxin (beta-BuTX) on cultured cerebellar granular neurons (CGN). Beta-BuTX had a potent time- and concentration-dependent neurotoxic effect on mature CGN. Beta-BuTX appeared to destroy initially the neurites and then caused neuronal death by both apoptotic and necrotic processes. Inspection using Nomarski optics showed that these neurons displayed morphological features of necrotic cells, including cell swelling, loss of membrane integrity and eventual dissolution of the cell. Staining with the fluorescent dye Hoechst 33258 showed that beta-BuTX-treated neuron bodies stained more densely with smaller apoptotic bodies. Using microspectrofluorimetry and fura-2 to measure cytosolic [Ca(2+)] ([Ca(2+)](i)), beta-BuTX markedly increased [Ca(2+)](i). BAPTA-AM, EGTA, MK 801 and diltiazem not only attenuated the beta-BuTX-mediated rise in [Ca(2+)](i) but also attenuated beta-BuTX-mediated neurotoxicity. In addition, these Ca(2+) inhibitors prevented the beta-BuTX-induced generation of reactive nitrogen species. The NO synthase inhibitor N(G)-methyl- l-arginine) also exhibited neuroprotection. This is the first report showing that beta-BuTX-induced CGN death is mediated, at least in part, by excessive generation of NO triggered by [Ca(2+)](i) overloading. Activation of NMDA receptors and L-type calcium channels is apparently involved in the increase in [Ca(2+)](i) induced by this neurotoxin. This potent neurotoxin will be a useful tool for studying neurotoxic processes and using this model system will allow us to find neuroprotective agents.