The cardiac Na⁺-Ca²⁺ exchanger (NCX) contributes to cellular injury during hypoxia, as its altered function is largely responsible for a rise in cytosolic [Ca²⁺]. In addition, the NCX in guinea-pig ventricular myocytes undergoes profound inhibition during hypoxia, and rapid reactivation during reoxygenation. The mechanisms underlying these changes in NCX activity are likely complex due to the participation of multiple inhibitory factors, including altered cytosolic [Na⁺], pH and ATP. Our main hypothesis is that oxidative stress is an essential trigger for rapid NCX reactivation in guinea-pig ventricular myocytes, and is thus a critical factor in determining the timing and magnitude of calcium overload. This hypothesis was evaluated in cardiac myocytes using fluorescent indicators to measure cytosolic [Ca²⁺] and oxidative stress. An NCX antisense oligonucleotide was used to decrease NCX protein expression in some experiments. Our results indicate that NCX activity is profoundly inhibited in hypoxic guineapig ventricular myocytes, but is reactivated within 1-2 minutes of reoxygenation, at a time of rising oxidative stress. We also found that several interventions to decrease oxidative stress, including antioxidants and diazoxide, prevented NCX reactivation and Ca²⁺ overload during reoxygenation. Furthermore, application of exogenous H₂O₂ was sufficient by itself to reactivate the NCX during sustained hypoxia, and could reverse the suppression of reoxygenation-mediated NCX reactivation by diazoxide. These data suggest that elevated oxidative stress in reoxygenated guinea-pig ventricular myocytes is required for rapid NCX reactivation, and thus reactivation should be viewed as an active process, rather than being due to the simple decline of NCX inhibition.