Hypoxic effects on glutamate uptake and ATP content in glial cells were investigated by using cultured C6 glioma cells. Mild regressive changes were found depending on the duration of the hypoxic insult, but necrosis or detachment of the cells from the substratum was rarely observed. Glutamate uptake was relatively well preserved after a short hypoxic insult, while a marked decrease in glutamate uptake was observed after hypoxia of long duration. The uptake of sucrose was reduced in a similar pattern to glutamate uptake. Hypoxic insult resulted in a significant reduction of the ATP content in glial cells. Therefore, the decrease in glutamate uptake by glial cells under hypoxia is likely to be due to ATP dependency, and not to the failure of a specific glutamate uptake system, but the failure of a general uptake of the glial cells owing to the energy-dependent membrane dysfunction by ATP depletion. These findings suggest that there are phased changes of astrocytic functions in a hypoxic condition, a preservative phase in the initial stages and then a dysfunctional phase in the later stages of hypoxia.