Reactive oxygen species production is necessary to induce cell death following hypoxia/reoxygenation but the effect of reactive oxygen species produced during hypoxia on mitochondrial permeability transition pore (mPTP) opening and cell death is not established. Here we designed a model of hypoxia/reoxygenation in isolated cardiomyocytes measuring simultaneously reactive oxygen species production, mPTP opening and cell death in order (i) to establish a causal relationship between them, and (ii) to investigate the roles of various reactive oxygen species in mPTP opening. The percentage of cardiomyocytes exhibiting mPTP opening during reoxygenation increased with the duration of hypoxia. Antioxidants increased the time to mPTP opening when present during hypoxia but not at reoxygenation. This was associated with a drop in hydroxyl radical and hydrogen peroxide during hypoxia and the first minutes of reoxygenation. The increase in time to mPTP opening was accompanied by an improvement in cell viability reflected by maintenance of superoxide production at reoxygenation. Cyclosporin A delayed both the time to mPTP opening and cell death despite maintenance of reactive oxygen species production during hypoxia. These findings demonstrate that reactive oxygen species production precedes mPTP opening and that reactive oxygen species produced during hypoxia, particularly hydroxyl radicals and hydrogen peroxide, are necessary to induce mPTP opening which depends on hypoxia duration.