Mitochondrial DNA (mtDNA) encodes critical subunit proteins of the oxidative phosphorylation (OXPHOS) complex that generates ATP. This study tested the hypothesis that mitochondrial gene expression in neural cells is regulated by energy demand, as modified via stimulation of cellular sodium transport. Exposure of PC12S cells to the sodium ionophore monensin (250 nm) for 1–6 h caused a 13–60% decrease in cellular ATP (from 15 to 5 nmol per mg protein at 6 h). Levels of mitochondrial DNA-encoded mRNAs (mt-mRNAs) increased significantly (150%) within the first hour of exposure to monensin, and then decreased significantly (50%) at 3–4 h. Levels of mtDNA-encoded 12S rRNA and nuclear DNA-encoded OXPHOS subunit mRNAs were not significantly affected. Exposure of primary cerebellar neuronal cultures to the excitatory amino acid glutamate caused a similar rapid and significant increase followed by a significant decrease in cell mt-mRNA levels. The monensin-induced initial increase in mt-mRNA levels was abolished by pretreatment with actinomycin D or by reducing extracellular sodium ion concentration. The monensin-induced delayed reduction in mt-mRNA levels was accelerated in the presence of actinomycin D, and was accompanied by a 67% reduction in the half-life (from 3.6 to 1.2 h). Exposure of PC12S cells to 2-deoxy-d-glucose significantly decreased cellular ATP levels (from 14.2 to 7.1 nmol per mg protein at 8 h), and increased mt-mRNA levels. These results suggest a physiological transcriptional mechanism of regulation of mitochondrial gene expression by energy demand and a post-transcriptional regulation that is independent of energy status of the cell.