Failure of glutamate uptake during ischemia can lead to neurotoxic accumulations of glutamate in brain extracellular space. Hypoxia and acidosis are metabolic consequences of ischemia that may individually or in combination impair glutamate uptake. We used primary rat astrocyte cultures to study the effects of acidosis, chemical hypoxia, and the combination of acidosis plus chemical hypoxia on glutamate uptake. Chemical hypoxia alone reduced uptake by 35-45%. Reduction in pH from 7.4 to 5.8 also caused a significant but incomplete inhibition of glutamate uptake, and this effect was more pronounced in medium buffered with CO2/bicarbonate. However, the combination of chemical hypoxia plus acidosis reduced glutamate uptake to below 10% of controls. Astrocyte ATP levels, like glutamate uptake, were significantly reduced by chemical hypoxia and further reduced by the combination of hypoxia plus acidosis. Acidosis under normoxic conditions had no significant effect on astrocyte ATP levels. These results suggest two mechanisms by which acidosis may contribute to failure of astrocyte glutamate uptake during ischemia: Acidosis may act in concert with hypoxia to cause ATP depletion, and acidosis may also have direct effects on glutamate transporters unrelated to effects on cellular ATP levels. pH effects on glutamate uptake may be an important factor affecting neuronal survival during incomplete ischemia.