It was recently proposed that in Jurkat cells, after inhibition of respiration by NO, glycolytically generated ATP plays a critical role in preventing the collapse of mitochondrial membrane potential (Δψ_m) and thus apoptotic cell death. We have investigated this observation further in primary cultures of rat cortical neurons and astrocytes—cell types that differ greatly in their glycolytic capacity. Continuous and significant (≈85%) inhibition of respiration by NO (1.4 μM at 175 μM O₂) generated by [(z)-1-[2-aminoethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2 diolate (DETA-NO) initially (10 min) depleted ATP concentrations by ≈25% in both cell types and increased the rate of glycolysis in astrocytes but not in neurons. Activation of glycolysis in astrocytes, as judged by lactate production, prevented further ATP depletion, whereas in neurons, which do not invoke this mechanism, there was a progressive decrease in ATP concentrations over the next 60 min. During this time, there was a persistent mitochondrial hyperpolarization and absence of apoptotic cell death in astrocytes, whereas in the neurons there was a progressive fall in Δψ_m and increased apoptosis. After glucose deprivation or treatment with inhibitors of the F₁F₀-ATPase and adenine nucleotide translocase, astrocytes responded to NO with a fall in Δψ_m and apoptotic cell death similar to the response in neurons. Finally, although treatment of astrocytes with NO partially prevented staurosporin-induced collapse in Δψ_m and cell death, NO and staurosporin synergized in decreasing Δψ_m and inducing apoptosis in neurons. These results demonstrate that although inhibition of cellular respiration by NO leads to neurotoxicity, it may also result in initial neuroprotection, depending on the glycolytic capacity of the particular cell.