Glutamate uptake is coupled to counter-transport of K+, and high external K+ concentrations can induce reversal of glutamate uptake in whole-cell patch-clamp and isolated membrane preparations. However, high external K+ causes little or no reversal of glutamate uptake in intact astrocytes, suggesting a regulatory mechanism not evident in membrane preparations. One mechanism by which intact cells could limit the effects of altered extracellular ion concentrations on glutamate transport is by compensatory changes in intracellular Na+ concentrations. This possibility was examined using astrocyte cultures treated in two ways to reduce the driving force for glutamate uptake: incubation in high K+ (with reciprocal reduction in Na+), and incubation with metabolic inhibitors to induce ATP depletion. ATP depletion produced a rise in intracellular Na+, a collapse of the membrane sodium gradient and a massive reversal of glutamate uptake. By contrast, incubation in high K+/low Na+ medium did not significantly alter the sodium gradient and did not induce glutamate uptake reversal. The sodium gradient was shown to be maintained under these conditions by compensatory reductions in intracellular Na+ that approximately matched the reductions in extracellular Na+. These findings suggest a mechanism by which astrocytes may limit reversal of glutamate uptake under high K+/low Na+ conditions, and further suggest a general mechanism by which Na(+)-dependent transport processes could be shielded from fluctuating extracellular ion concentrations.