Na(+)-Ca2+ exchange has been identified as a mechanism for regulation of intracellular Ca ion concentration ([Ca2+]i) in neurons of invertebrates and vertebrates, but for mammalian central neurons its role in restoration of resting [Ca2+]i after transient increases induced by stimulation has been less clear. We have examined the recovery of [Ca2+]i following K+ depolarization and glutamate receptor activation of cultured mouse hippocampal neurons using the Ca(2+)- sensitive dye Fura-2. Reduction of the transmembrane Na+ gradient by removal of external Na+ slowed the recovery of neurons from imposed Ca2+ loads. We observed that [Ca2+]i regulation was disrupted more severely when N-methyl-D-glucamine (N-MG), Tris, or choline rather than Li+ replaced external Na+. Additional disruption of intracellular pH regulation by substitutes other than Li+ may account for this difference. Measurement of [Ca2+]i and [H+]i (using the H(+)-sensitive dye BCECF) during glutamate receptor activation indicated that Ca2+ influx resulted in production of intracellular H+, and that Li+ but not N-MG could prevent cytoplasmic acidification on removal of external Na+. We also observed that intracellular acidification alone was sufficient to slow recovery from Ca2+ load. We conclude, therefore, that Na(+)-Ca2+ exchange contributes to recovery of [Ca2+]i after stimulation leading to Ca2+ entry into hippocampal neurons, and that Na(+)-H+ exchange limits the acidification (and secondary increase in [Ca2+]i) that accompanies Ca2+ influx. We suggest that because both Na(+)-Ca2+ and Na(+)-H+ exchangers will be compromised during ischemia and hypoglycemia, increased intracellular H+ may synergize with cytoplasmic Ca2+ to potentiate excitotoxic neuronal death.