The involvement of brain lactate in neuronal metabolism was analyzed by ex vivo NMR spectroscopy with rats under the effects of pentobarbital, αchloralose or morphine, which were infused with a solution of either [1‐¹³C]glucose+lactate or glucose+[3‐¹³C]lactate for 20 min. Electroencephalogram recordings indicated different brain electrical activity levels under the three drugs with a clear distinction between pentobarbital, on the one hand, and αchloralose and morphine on the other. Labeling of metabolites in brain perchloric acid extracts and of blood glucose and lactate was determined by ¹³C‐ and/or ¹H‐observed/¹³C‐edited‐NMR spectroscopy. The following were found: (i) the ratio between glutamate C3 and C4 ¹³C‐enrichments increased from pentobarbital to αchloralose and morphine whatever the labeled precursor, indicating a link between metabolic and electrical activity; (ii) under glucose+[3‐¹³C]lactate infusion, alanine C3 and acetyl–CoA C2 enrichments were higher than that of lactate C3, revealing the occurrence of an isotopic dilution of the brain exogenous lactate (arising from blood) by lactate from brain (endogenous lactate); the latter was synthesized from glycolysis in a compartment other than the neurons; (iii) the contributions of labeled glucose and lactate to acetyl–CoA C2 enrichment indicated that the involvement of blood glucose relative to that of blood lactate to brain metabolism was correlated with brain activity. It can therefore be concluded that the brain electrical activity‐dependent increase in the contribution of blood glucose relative to that of blood lactate to brain metabolism occurred partly via the increase in the metabolism of lactate generated from astrocytic glycolysis. This conclusion supports the hypothesis of an astrocyte–neuron lactate shuttle component in the coupling mechanism between cerebral activity and energy metabolism. Copyright © 2003 John Wiley & Sons, Ltd.