The potential of protease inhibitors E-64, calpain inhibitor I (CPI-I) and MDL28170 to protect hippocampal neurons in an in vitro model of neurotoxicity was investigated. Hippocampal cultures were treated with glutamate, and neurotoxicity was quantified. Glutamate treated cultures exhibited damage to approximately 50% of neurons. In contrast only 20-30% of neurons were damaged in cultures treated with glutamate and calpain inhibitors. E-64 and CPI-I are capable of protecting neurons from injury only in pre-treatment schedule. MDL28170 exhibits a neuroprotective effect in the pre-treatment schedule and also even when given immediately after the cultures had been switched to the glutamate-containing medium. Although the neuroprotective effect of MDL28170 in the postreatment schedule was modest, this supports a strick link between the ability of protease inhibitors to penetrate cellular membranes and their potency of neuroprotection. These data provide evidence that calpain-induced proteolysis is an important pathogenic factor in brain injury and suggest that calpain inhibitors may be considered as a powerful mean to counteract the sequelea of neurotoxicity.