The neurotoxicity induced by depolarization with high-K + was investigated in rat hippocampal organotypic slice cultures. The exposure of cultures to 90 mM K + solution for 30 min caused a severe neuronal injury in CA1 region while less damage was observed in CA3 and dentate gyrus over the following day. This neurotoxicity was prevented in a concentration dependent manner by NMDA antagonist MK-801 or CPP. Non-NMDA antagonist, DNQX, had no protective effect. Omission of Ca 2 + from the exposure solution prevented the neurotoxicity. Voltage-dependent Ca 2 + channel blockers, nifedipine and flunarizine, failed to prevent the neurotoxicity. These results suggest that the Ca 2 + influx through the NMDA receptor is predominantly involved in this neurotoxicity. Apparent tissue swelling was observed immediately after the depolarization. This swelling was completely inhibited by omission of Cl - from the exposure solution, accompanied with complete protection against neurotoxicity. This suggests that Cl - -dependent tissue swelling also largely contributes to the neurotoxicity. Depolarization with application of MK-801 (10 μM) or omission of Ca 2 + from the solution still caused apparent swelling, despite these treatment protected neuronal death. We hypothesize that Cl - -dependent tissue swelling may be involved in the release of the excitatory amino acid, which activates the NMDA receptor.