Glutamate uptake into astrocytes and the resulting increase in intracellular Na+ (Na+(i)) have been identified as a key signal coupling excitatory neuronal activity to increased glucose utilization. Arguments based mostly on mathematical modeling led to the conclusion that physiological concentrations of glutamate more than double astrocytic Na+/K+-ATPase activity, which should proportionally increase its ATP hydrolysis rate. This hypothesis was tested in the present study by fluorescence monitoring of free Mg2+ (Mg2+(i)), a parameter that inversely correlates with ATP levels. Glutamate application measurably increased Mg2+(i) (i.e. decreased ATP), which was reversible after glutamate washout. Na+(i) and ATP changes were then directly compared by simultaneous Na+(i) and Mg2+ imaging. Glutamate increased both parameters with different rates and blocking the Na+/K+-ATPase during the glutamate-evoked Na+(i) response, resulted in a drop of Mg2+(i) levels (i.e. increased ATP). Taken together, this study demonstrates the tight correlation between glutamate transport, Na+ homeostasis and ATP levels in astrocytes.