Although tight regulation of intracellular pH (pHi) is critical for the survival under stress, paradoxically a slowed recovery of phi under hypoxic injury may be cardioprotective. In this study, we investigated the recovery of phi after hypoxia-induced intracellular acidosis in cardiomyocytes loaded with the H+-sensitive dye SNARF-1. Exposure of single cardiomyocytes to 2,4- dinitrophenol (DNP), an inhibitor of mitochondrial oxidative phosphorylation, induced significant intracellular acidification. However, within 10-12 min upon removal of DNP, cardiomyocytes restituted their intracellular H+ concentration. The presence either of 5-N-ethyl-N-isopropylamiloride (EIPA) an inhibitor of Na/H antiporter, or 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid (DIDS), an inhibitor of bicarbonate-dependent exchange, did not modify the cellular response to DNP. But, combined use of EIPA and DIDS prevented the restitution of intracellular pH following removal of DNP. This study, thus, demonstrated, for the first time, that blockade of both Na/H and bicarbonate-dependent exchange is necessary and sufficient to maintain the hypoxia-induced intracellular acidification. Therefore, concomitant blockade of both pH-regulating mechanisms deserves to be further considered as a novel strategy against hypoxia-reoxygenation injury in the heart.