Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K⁺ as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of , a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to and in the somatosensory cortex of anesthetized mice in response to i.v. . Unexpectedly, had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes.