In glucose-deprived cerebellar granule cells, substitution of extracellular Na+ with Li+ or Cs+ prevented N-methyl-D-aspartate (NMDA)-induced excitotoxicity. NMDA stimulated 45Ca2+ accumulation and ATP depletion in a Na-dependent manner, and caused neuronal death, even if applied while Na,K-ATPase was inhibited by 1 mM ouabain. The cells treated with NMDA in the presence of ouabain accumulated sizable 45Ca2+ load but most of them failed to elevate cytosolic [Ca2+] upon mitochondrial depolarization. Na/Ca exchange inhibitor, KB-R7943, inhibited Na-dependent and NMDA-induced 45Ca2+ accumulation but only if Na,K-ATPase activity was compromised by ouabain. In cells energized by glucose and exposed to NMDA without ouabain, KB-R7943 reduced NMDA-elicited ionic currents by 19% but failed to inhibit 45Ca2+ accumulation. It appears that a large part of NMDA-induced Ca2+ influx in depolarized and glucose-deprived cells is mediated by reverse Na/Ca exchange. A high level of reverse Na/Ca exchange operation is maintained by a sustained Na+ influx via NMDA channels and depolarization of the plasma membrane. In cells energized by glucose, however, most Ca2+ enters directly via NMDA channels because Na,K-ATPase regenerating Na+ and K+ concentration gradients prevents Na/Ca exchange reversal. Since under these conditions Na/Ca exchange extrudes Ca2+, its inhibition destabilizes Ca2+ homeostasis.