This study investigated the selective effects of intracellular (pHi) or extracellular change in pH on reoxygenation-induced Ca2+ overload in simulated myocardial hypoxia. Experiments were performed in cultured cardiomyocytes isolated from the ventricle of neonatal ICR mouse. A model of chemical hypoxia with 2 mM sodium cyanide was developed to mimic the ATP depletion of hypoxia. This chemical hypoxia was "reoxygenated" and the dynamics in intracellular Ca2+ concentration ([Ca2+]i) and pHi were monitored using the fluorescent dyes fura-2 and 2', 7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. During a 40-min chemical hypoxia, pHi progressively fell from 7.2 to 6.6. Reoxygenation with control solution caused rapid recovery of pHi and a marked increase in [Ca2+]i (1884 +/- 136 nM). Intracellular acidotic reoxygenation produced by lactate apparently prolonged the time course of pHi recovery and significantly suppressed reoxygenation-induced Ca2+ overload (1170 +/- 118 nM, P = 0.008). Extracellular acidotic reoxygenation with 2-(N-morpholino) ethanesulfonic acid (pK = 5.96) buffer somewhat suppressed the Ca2+ overload; however, the maximal value of [Ca2+]i was not reduced significantly compared with the control (1790 +/- 122 nM, P = 0.130). In addition, inhibition of Na(+)-H+ exchange by amiloride potentiated prolongation of intracellular acidosis during reoxygenation and resulted in a minimal increase in [Ca2+]i (985 +/- 102 nM, P = 0.004). These results suggest that reoxygenation-induced Ca2+ overload is closely correlated with intracellular pH in the initial phase of reoxygenation, and the protective effects of extracellular acidosis is principally mediated by intracellular acidification of reoxygenated cardiomyocytes.