Oxidative stress and disrupted energy metabolism are common to many pathological conditions of the brain. Because astrocytes play an important role in the glucose metabolism of the brain, we have investigated whether sustained oxidative stress affects astroglial glucose metabolism with cultured primary rat astrocytes as a model system. Cultured astrocytes were exposed to a sustained concentration of approximately 50 μM H2O2 in the presence of [U-13C]glucose, and cellular and extracellular contents of lactate and glucose were analysed by enzymatic assays and NMR spectroscopy. Exposure of the cells to sustained H2O2 stress for up to 120 min significantly lowered the rate of lactate accumulation in the media to 61% ± 14% of that in cultures incubated without peroxide. In addition, the ratio of lactate release to glucose consumption was lowered in peroxide-treated astrocytes to 77% ± 13% of that in control cells, and the specific activity of glyceraldehyde-3-phosphate dehydrogenase had declined to about 10% of control cells within 90 min. In addition, the 13C enrichment of intracellular and extracellular [13C]lactate was about 30% and 95%, respectively, and was not affected by the presence of peroxide, demonstrating that two metabolic pools of lactate are present in cultured astrocytes. The decreased rate of lactate production by astrocytes that have been exposed to peroxide stress is a new example of an alteration by oxidative stress of an important metabolic pathway in astrocytes. Such alterations could contribute to the pathological conditions that have been connected with oxidative stress and disrupted energy metabolism in the brain. © 2009 Wiley-Liss, Inc.