The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na+-dependent transporter. Evidence had been provided that Na+,K+-ATPase might be involved in this process. We have now measured the activity of Na+,K+-ATPase in cultured astrocytes, using ouabain-sensitive 86Rb uptake as an index. L-Glutamate increases glial Na+,K+-ATPase activity in a concentration-dependent manner with an EC50 = 67 microM. Both L- and D-aspartate, but not D-glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na+,K+-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain (K0.5 = 113 microM), compatible with the presence of an alpha1 isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain (K0.5 = 20 nM), thus revealing a high-affinity site akin to the alpha2 isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na+,K+-ATPase that can be mobilized in response to increased neuronal activity.