During myocardial ischemia, a large reduction of tissue pH develops, and tissue pH returns to normal after reperfusion. In recent studies, we evaluated the role of pH in ischemia/reperfusion injury to cultured cardiac myocytes and perfused papillary muscles. Acidosis (pH < or = 7.0) protected profoundly against cell death during ischemia. However, the return from acidotic to normal pH after reperfusion caused myocytes to lose viability. This worsening of injury is a 'pH paradox' and was mediated by changes of intracellular pH (pH(i)), since manipulations that caused pH(i), to increase more rapidly after reperfusion accelerated cell killing, whereas manipulations that delayed the increase of pH(i) prevented loss of myocyte viability. Specifically, inhibition of the Na+/H+ exchanger with dimethylamiloride or HOE694 delayed the return of physiologic pH(i) after reperfusion and prevented reperfusion-induced cell killing to both cultured myocytes and perfused papillary muscle. Dimethylamiloride and HOE694 did not reduce intracellular free Ca2+ during reperfusion. By contrast, reperfusion with dichlorobenzamil, an inhibitor of Na+/Ca2+ exchange, decreased free Ca2+ but did not reduce cell killing. Thus, the pH paradox is not Ca(2+)-dependent. Our working hypothesis is that ischemia activates hydrolytic enzymes, such as phospholipases and proteases, whose activity is inhibited at acidotic pH. Upon reperfusion, the return to normal pH releases this inhibition and hydrolytic injury ensues. Increasing pH(i) may also induce a pH-dependent mitochondrial permeability transition and activate the myofibrillar ATPase, effects that increase ATP demand and compromise ATP supply. In conclusion, acidotic pH is generally protective in ischemia, whereas a return to physiologic pH precipitates lethal reperfusion injury to myocytes.