We used isolated myocytes to investigate the role of mitochondrial re-energization and Ca 2+ influx during reperfusion on hypercontracture, loss of Ca 2+ homeostasis and contractile function.Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion injury was assessed from hypercontracture, loss of Ca 2+ homeostasis ([Ca 2+ ] i measured with fura-2) and failure of contraction in response to electrical stimulation. Mitochondrial membrane potential was followed using the potentiometric dye tetramethylrhodamine ethyl ester.Metabolic inhibition led to contractile failure and rigor accompanied by a sustained increase in [Ca 2+ ] i . Reperfusion after 10 min metabolic inhibition led to an abrupt repolarization of the mitochondrial membrane potential (after 25.5±1.2 s), a transient fall in [Ca 2+ ] i followed by an abrupt hypercontracture (37.1±1.8 s) in 84% of myocytes. Ca 2+ homeostasis (diastolic [Ca 2+ ] i <250 nM) recovered in only 23.3±5.1% of cells and contractions recovered in 15.3±2.2%. Oligomycin abolished the hypercontracture on reperfusion, but mitochondrial repolarization was unaffected. Preventing Ca 2+ influx during reperfusion with Ca 2+ -free Tyrode or with an inhibitor of Na + /Ca 2+ exchange did not prevent the hypercontracture, but increased the percentage of cells recovering Ca 2+ homeostasis and contractile function. The presence of 0.5 μM cyclosporin A did not prevent hypercontracture but increased the percentage of cells recovering Ca 2+ homeostasis to 56.2±3.6% and contractile function to 52±4.3%.Reperfusion-induced hypercontracture, and loss of Ca 2+ homeostasis and contractile function are initiated following mitochondrial re-energization. The hypercontracture requires the production of oxidative ATP but not Ca 2+ influx during reperfusion. Loss of Ca 2+ homeostasis and contractile function are linked to Ca 2+ influx during reperfusion, probably via opening of mitochondrial permeability transition pores.