To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During anoxia, intracellular pH (pH_i) decreased to 6.3, mitochondria depolarized, and ATP decreased to <1% of basal values, but the mitochondrial permeability transition (MPT) did not occur as assessed by confocal microscopy from the redistribution of cytosolic calcein into mitochondria. Moreover, cell viability remained >90%. After reperfusion at pH 7.4, pH_i returned to pH 7.2, the MPT occurred, and most hepatocytes lost viability. In contrast, after reperfusion at pH 6.2 or with Na⁺-free buffer at pH 7.4, pH_i did not rise and cell viability remained >80%. After acidotic reperfusion, the MPT did not occur. When hepatocytes were reperfused with cyclosporin A (0.5–1 μM) at pH 7.4, the MPT was prevented and cell viability remained >80%, although pH_i increased to 7.2. Reperfusion with glycine (5 mM) also prevented cell killing but did not block recovery of pH_i or the MPT. Retention of cell viability was associated with recovery of 30–40% of ATP. In conclusion, preventing the rise of pH_i after reperfusion blocked the MPT, improved ATP recovery, and prevented cell death. Cyclosporin A also prevented cell killing by blocking the MPT without blocking recovery of pH_i. Glycine prevented cell killing but did not inhibit recovery of pH_i or the MPT.