Dehydroevodiamine has been reported to have neuroprotective and antiamnesic effects. This study examined the effects of dehydroevodiamine on glutamate release and uptake in cultured cerebellar cells. Chronic dehydroevodiamine exposure decreased the viability of granule cells. The basal and N-methyl-D-aspartate (NMDA)-induced release of glutamate from granule cells were decreased (26 and 14%) by dehydroevodiamine. The NMDA-induced release of glutamate was concentration-dependently inhibited in the granule cells. The basal and NMDA-induced releases of glutamate in chronically dehydroevodiamine-preexposed granule cells were unaffected by dehydroevodiamine. Glutamate uptake in the glial cells incubated without and with cAMP was inhibited (31% and 8%, respectively) by dehydroevodiamine. In the chronically dehydroevodiamine-preexposed glial cells, glutamate uptake was increased (8%) in the cAMP-coexposed glial cells by dehydroevodiamine but was unaffected in the naive cells. In addition, dehydroevodiamine potentiated (from 20% to 34%) the inhibition of L-pyrollidine-2,4-dicarboxylic acid (PDC) on glutamate uptake in naive glial cells, but this inhibition was reduced (from 41% to 26%) in cAMP-coexposed glial cells. These results suggest that dehydroevodiamine inhibits glutamate uptake and release. Furthermore, the results suggest that the characteristics of glutamate release and uptake in granule and glial cells may be altered by chronic exposure to dehydroevodiamine.