OBJECTIVE - To investigate the effect of acute insulin-induced hypoglycemia on cerebral glucose metabolism in healthy humans, measured by 13C magnetic resonance spectroscopy (MRS). RESEARCH DESIGN AND METHODS - Hyperinsulinemic glucose clamps were performed at plasma glucose levels of 5 mmol/L (euglycemia) or 3 mmol/L (hypoglycemia) in random order in eight healthy subjects (four women) on two occasions, separated by at least 3 weeks. Enriched [1-13C]glucose 20% w/w was used for the clamps to maintain stable plasma glucose labeling. The levels of the 13C-labeled glucose metabolites glutamate C4 and C3 were measured over time in the occipital cortex during the clamp by continuous 13C MRS in a 3T magnetic resonance scanner. Time courses of glutamate C4 and C3 labeling were fitted using a one-compartment model to calculate metabolic rates in the brain. RESULTS - Plasma glucose 13C isotopic enrichment was stable at 35.± 1.8% during euglycemia and at 30.2 ± 5.5% during hypoglycemia. Hypoglycemia stimulated release of counterregulatory hormones (all P < 0.05) and tended to increase plasma lactate levels (P = 0.07). After correction for the ambient 13C enrichment values, label incorporation into glucose metabolites was virtually identical under both glycemic conditions. Calculated tricarboxylic acid cycle rates (VTCA) were 0.48 ± 0.03 μmol/g/min during euglycemia and 0.43 ± 0.08 μmol/g/min during hypoglycemia (P = 0.42). CONCLUSIONS - These results indicate that acute moderate hypoglycemia does not affect fluxes through the main pathways of glucose metabolism in the brain of healthy nondiabetic subjects.