<p>ROS overproduction is an important contributor to delayed ischemia/reperfusion induced neuronal injury, but relevant mechanisms remain poorly understood. We used oxygen–glucose deprivation (OGD)/reperfusion in mouse hippocampal slices to investigate ROS production in the CA1 pyramidal cell layer during and after transient ischemia. OGD evoked a 2-stage increase in ROS production: 1st—an abrupt increase in ROS generation starting during OGD followed by a marked slowing; and 2nd—a sharp ROS burst starting ~ 40&#xa0;min after reperfusion. We further found that a slight mitochondrial hyperpolarization occurs shortly after OGD termination. Consequently, we showed that administration of low dose FCCP or of FTY720 (both of which cause mild, ~ 10%, mitochondrial depolarization), markedly diminished the delayed ROS burst, suggesting that mitochondrial hyperpolarization contributes to ROS production after reperfusion. Zn²⁺ chelation after OGD withdrawal also substantially decreased the late surge of ROS generation—in line with our prior studies indicating a critical contribution of Zn²⁺ entry into mitochondria via the mitochondrial Ca²⁺ uniporter (MCU) to mitochondrial damage after OGD. Thus, reperfusion-induced mitochondria hyperpolarization and mitochondrial Zn²⁺ accumulation both contribute to mitochondrial ROS overproduction after ischemia. As these events occur after reperfusion, they may be amenable to therapeutic interventions.</p>