During hypoglycemia, substrates other than glucose have been suggested to serve as alternate neural fuels. We evaluated brain uptake of endogenously produced lactate, alanine, and leucine at euglycemia and during insulin-induced hypoglycemia in 17 normal subjects. Cross-brain arteriovenous differences for plasma glucose, lactate, alanine, leucine, and oxygen content were quantitated. Cerebral blood flow (CBF) was measured by Fick methodology using N₂O as the dilution indicator gas. Substrate uptake was measured as the product of CBF and the arteriovenous concentration difference. As arterial glucose concentration fell, cerebral oxygen utilization and CBF remained unchanged. Brain glucose uptake (BGU) decreased from 36.3 ± 2.6 to 26.6 ± 2.1 μmol·100 g of brain⁻¹·min⁻¹ (P < 0.001), equivalent to a drop in ATP of 291 μmol·100 g⁻¹·min⁻¹. Arterial lactate rose (P < 0.001), whereas arterial alanine and leucine fell (P < 0.009 and P < 0.001, respectively). Brain lactate uptake (BLU) increased from a net release of −1.8 ± 0.6 to a net uptake of 2.5 ± 1.2 μmol·100 g⁻¹·min⁻¹ (P < 0.001), equivalent to an increase in ATP of 74 μmol·100 g⁻¹·min⁻¹. Brain leucine uptake decreased from 7.1 ± 1.2 to 2.5 ± 0.5 μmol·100 g⁻¹·min⁻¹ (P < 0.001), and brain alanine uptake trended downward (P < 0.08). We conclude that the ATP generated from the physiological increase in BLU during hypoglycemia accounts for no more than 25% of the brain glucose energy deficit.