Neuronal damage associated with in vivo hypoglycemia has been suggested to be excitotoxic due to the release of excitatory amino acids and the protective effect of glutamate receptor antagonists. The production of reactive oxygen species (ROS) has been also implicated in hypoglycemic damage. Excitotoxicity involves oxidative stress, insofar as the influx of calcium through N-methyl-D-aspartate (NMDA) receptors stimulates ROS production. We have studied the participation of NMDA receptors and intracellular calcium in ROS production and cell death triggered during moderate and severe glycolysis inhibition in cultured hippocampal neurons. Iodoacetate (IOA), an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), dose dependently reduces ATP levels and cell survival and increases the intracellular concentration of calcium. During mild glycolysis inhibition, the increases in intracellular calcium, ROS production, and cell death are dependent on NMDA receptor activation. In contrast, during severe glycolysis impairment, these processes are not inhibited by NMDA receptor blockade. BAPTA-AM and vitamin E efficiently reduce ROS generation and cell death under both conditions. Results suggest that calcium influx through NMDA receptors is involved in ROS production and neuronal damage resulting from moderate energy depletion, whereas intracellular calcium increase and ROS generation during severe glycolysis inhibition are more related to energy depletion.