The hypothesis was tested that oxidative metabolism, mainly fueled by glutamate itself, provides the energy for active, Na(+),K(+)-ATPase-catalyzed Na(+) extrusion following glutamate uptake in conjunction with Na(+). This hypothesis was supported by the following observations: (i) glutamate had either no effect or caused a slight reduction in glycolytic rate, measured as deoxyglucose phosphorylation; (ii) D-aspartate, which is accumulated by the L-glutamate carrier, but cannot be metabolized by the cells, caused an increase in glycolytic rate; (iii) monensin which, like D-aspartate, stimulates the intracellular, Na(+)-activated site of the Na, K-ATPase and thus energy metabolism, but provides no metabolic substrate, stimulated both glycolysis and glucose oxidation; and (iv) oxidation of glucose was potently inhibited by glutamate, although glutamate is known to stimulate oxygen consumption in primary cultures of astrocytes, a combination showing that oxidation of a non-glucose substrate is increased in the presence of glutamate. These findings should be considered in attempts to understand metabolic interactions between neurons and astrocytes and regulation of energy metabolism in brain.