It has been postulated that lactate released from astrocytes may be the preferred metabolic substrate for neurons, particularly during intense neuronal activity (the astrocyte-neuron lactate shuttle hypothesis). We examined this hypothesis by exposing rat hippocampal slices to artificial cerebrospinal fluid containing either glucose or lactate and either N-methyl-D-aspartate, which activates neurons without stimulating astrocytic glucose uptake, or alpha-cyano-4-hydroxycinnamate, which blocks monocarboxylate transport across plasma and mitochondrial membranes. When exposed to N-methyl-D-aspartate, slices lost synaptic transmission and K+ homeostasis more slowly in glucose-containing artificial cerebrospinal fluid than in lactate-containing artificial cerebrospinal fluid. After N-methyl-D-aspartate exposure, slices recovered synaptic transmission more completely in glucose. These results suggest that hippocampal neurons can use glucose more effectively than lactate when energy demand is high. In experiments with alpha-cyano-4-hydroxycinnamate, 500 microM alpha-cyano-4-hydroxycinnamate caused loss of K+ homeostasis and synaptic transmission in hippocampal slices during normoxia. When 200 microM alpha-cyano-4-hydroxycinnamate was used, synaptic activity and intracellular pH in slices decreased significantly during normoxia. These results suggest that alpha-cyano-4-hydroxycinnamate may have blocked mitochondrial oxidative metabolism along with lactate transport. Thus, studies using alpha-cyano-4-hydroxycinnamate to demonstrate the presence of a lactate shuttle in the brain tissue may need reevaluation. Our findings, together with observations in the literature that (1) glucose is available to neurons during activation, (2) heightened energy demand rapidly activates glycolysis in neurons, and (3) activation of glycolysis suppresses lactate utilization, suggests that glucose is the primary substrate for neurons during neuronal activation and do not support the astrocyte-neuron lactate shuttle hypothesis.