Tert-butyl hydroperoxide (t-BuOOH) induces the mitochondrial permeability transition (MPT) in hepatocytes, leading to cell death. Using confocal microscopy, we visualized pyridine nucleotide oxidation and reactive oxygen species (ROS) formation induced by t-BuOOH. Reduced mitochondrial pyridine nucleotides (NADH and NADPH) were imaged by autofluorescence. Mitochondrial membrane potential, ROS, onset of MPT, and cell death were monitored with tetramethylrhodamine methyl ester (TMRM), dichlorofluorescin, calcein, and propidium iodide, respectively. t-BuOOH rapidly oxidized mitochondrial NAD(P)H. Oxidation was biphasic, and the second slower phase occurred during mitochondrial ROS generation. Subsequently, MPT took place, mitochondria depolarized, and cells died. beta-Hydroxybutyrate, which reduces mitochondrial NAD+, delayed cell killing, but lactate, which reduces cytosolic NAD+, did not. Trifluoperazine, which inhibits MPT, did not block the initial oxidation of NAD(P)H but prevented the second phase of oxidation, partially blocked ROS formation, and preserved cell viability. The antioxidants, deferoxamine and diphenylphenylenediamine, also prevented the second phase of NAD(P)H oxidation. They also blocked ROS formation nearly completely and stopped cell killing. Both antioxidants also prevented the mitochondrial permeability transition and subsequent mitochondrial depolarization. In conclusion, NAD(P)H oxidation and ROS formation are critical events promoting MPT in oxidative injury and death of hepatocytes.