There was a large release of endogenous glutamate and of pre-accumulated [3H]-D-aspartate from rat hippocampal slices during deprivation of oxygen and glucose (in vitro ischemia). The role of Na(+)-dependent glutamate transporters in this process was investigated. The release of both glutamate and [3H]-D-aspartate was largely blocked by two competitive substrate analogues of the Na(+)-dependent glutamate transporters (L-trans-pyrrolidine-2,4-dicarboxylate and D,L-threo-B-hydroxyaspartate) if the substrate analogues were intracellularly loaded prior to the ischemia. The pre-loaded analogue, D,L-threo-B-hydroxyaspartate, did not block exocytotic release of glutamate, induced by high-potassium. Dihydrokainate, an inhibitor of a subset of the Na(+)-dependent transporters, did not inhibit ischemia-induced release of glutamate or [3H]-D-aspartate. However, it did block release induced by veratridine, which was also blocked by the pre-loaded substrate analogues. Dihydrokainate could still inhibit veratridine-induced release during ischemia, showing that conditions during ischemia did not reduce its efficacy. It is concluded that release of glutamate during ischemia is largely via reversal of the Na(+)-dependent glutamate transport system. The differential effects of dihydrokainate and the competitive substrate analogues on ischemia-induced release indicate that this release occurs via a subset of the glutamate transporters that are present in the hippocampus.