We have previously reported that the spontaneous release of [³H]5-hydroxytryptamine(5-HT) was markedly decreased by hypoxic insult in rat hippocampal slices. In the present study, the effect of glucose on 5-HT release was examined. Fractional release of [³H]5-HT was measured from an incubation medium exchanged every 10 min for 140 min and after stabilization of [³H]5-HT release, 10 or 20 min period of hypoxia was induced by exchanging the media which have been previously saturated by 95% N₂/5% CO₂ gas. In the media containing 1, 2, 5, 10 or 20 mM glucose, [³H]5-HT release was stabilized after 40 min of incubation. Exposure to hypoxia decreased [³H]5-HT release up to 60% of the control level in a glucose concentration-dependent manner and recovered gradually after hypoxic periods. However, in the media containing no glucose, the spontaneous release of [³H] 5-HT increased continuously during incubation. Moreover, when hippocampal slices were exposed to hypoxia, the [³H]5-HT release increased up to 150% of the control level and recovered gradually to the control level after hypoxic periods. These results demonstrate that hypoxia inhibits or enhances 5-HT release in the presence or absence of extracellular glucose respectively and suggest that the availability of extracellular glucose is a key factor to determine the direction of 5-HT release under hypoxic condition.