Pyruvate and malate (P/M) increase the contribution of mitochondria to neuronal calcium homeostasis. We have now found that cortical neuronal cultures utilize pyruvate preferentially over glucose. The supply of pyruvate and malate protects hippocampal and cortical neurons against delayed cell death occurring 24 h after glutamate exposure. High [Ca2+]i levels attained during and after glutamate exposure were reduced when neurons were incubated in the presence of P/M. At the single cell level, this was reflected in a decrease in the number of neurons that respond to glutamate with high rises in [Ca2+]i. The results suggest that the ability to prevent large increases in [Ca2+]i may underlie the beneficial effects of pyruvate and malate during glutamate excitotoxicity.