Uncertainty reigns over whether or not glutamate uptake in astrocytes leads to strong stimulation of glucose utilization, measured as accumulation of radioactive deoxyglucose-6-phosphate. This is an important issue, not only because glutamate is the major excitatory transmitter, but also because it has been postulated that glutamate-induced stimulation of glycolysis links brain excitation with activation of energy production. The effect of glutamate on deoxyglucose utilization in cultured rat and mouse astrocytes grown in different media and incubated under various conditions during the deoxyglucose assay has, therefore, been studied. Under most conditions, no stimulation occurred but rather a decrease in deoxyglucose utilization during exposure to glutamate; under certain conditions, the contribution of non-metabolized deoxyglucose to the intracellular 14C signal was significant.