To determine whether calmodulin plays a role in neurodegeneration after ischemia, effects of the selective calmodulin inhibitors calmidazolium and W7 were studied in organotypic cultures of rat hippocampus. Protection of pyramidal cells in the CA1 region of the hippocampus by calmidazolium and W7 against hypoxia/hypoglycemia suggests that activation of intracellular calmodulin plays a significant role in ischemic neuronal injury. Both ryanodine and TMB-8, inhibitors of intracellular Ca2+ release, failed to prevent ischemic neuronal injury. These results indicate that calmodulin, a major intracellular Ca2+ binding protein, plays a significant role in experimental ischemia-induced hippocampal neuronal injury in vitro.