We studied the effect of external pH (pHe) on cell injury, ATP content, and intracellular concentration of Ca2+ ([Ca2+]i), Na+ ([Na+]i), and H+ (pHi) during metabolic inhibition (NaCN + 2-deoxyglucose) in neonatal rat cardiomyocytes. Cell death during metabolic inhibition decreased at pHe < 7.4, with almost no cell death at pHe 6.0. Lowering pHe resulted in only temporary ATP conservation. During metabolic inhibition at pHe 7.4, [Ca2+]i rose from 86 +/- 44 nM to 2.5 +/- 0.4 microM, but at pHe 6.0, [Ca2+]i rose to only 510 +/- 215 nM. During metabolic inhibition at pHe 7.4, pHi decreased from 7.25 +/- 0.06 to 6.82 +/- 0.16, but at pHe 6.0, pHi decreased to 6.34 +/- 0.17. During metabolic inhibition at pHe 7.4, [Na+]i increased from 9.1 +/- 0.86 to 26.1 +/- 4.1 mM. At pHe 6.0, [Na+]i rose more rapidly, to 27.3 +/- 3.5 mM. At pHe < 7.4, sarcolemmal Na+/Ca2+ exchanger activity, involved in the development of Ca2+ overload, was decreased, as assessed during Na(+)-free incubation. We conclude that low pHe protects cardiomyocytes during metabolic inhibition by limiting Ca2+ overload via Na+/Ca2+ exchanger inhibition.