Excessive activation of glutamate receptors is neurotoxic, contributing to brain injury caused by cerebral ischemia. The pharmacology of glutamate neurotoxicity is difficult to study in animals because it is efficiently cleared from the extracellular space by a family of glutamate transporters. We have investigated the receptor specificity of endogenous glutamate's toxic effects in organotypic cultures of the hippocampus by acute blockade of these transporters. The organotypic cultures used in these transporters. The organotypic cultures used in these experiments preserve the intrinsic connections and regional differentiation of the hippocampus in long term culture and may more closely reproduce the pharmacology of the mature brain region. Membrane injury was measured with digital fluorescence imaging of the vital dye, propidium iodide, 24 h after a 30-min exposure to glutamate receptor agonists or to antagonists of glutamate transport. Confirming our previous results, bath-applied, exogenous glutamate caused dose-dependent neuronal injury. Glutamate was less potent than the selective agonists NMDA, AMPA, and quisqualate. Blockade of glutamate transport with the selective antagonists threo-hydroxy-aspartate and pyrrolidine-dicarboxylic acid also caused dose-dependent neuronal injury. Endogenous or exogenous glutamate toxicity was caused by a coactivation of both NMDA and AMPA/kainate receptors; blockade of either was sufficient to substantially prevent neuronal injury. Protective effects of combined application of antagonists were generally less than additive. We conclude that AMPA/kainate receptors play a more prominent role in glutamate neurotoxicity in organotypic cultures than in dissociated cortical or hippocampal cultures, acting together with NMDA receptors to cause neuronal injury.