During hypoxia of isolated cardiomyocytes, Ca 2 + entry into mitochondria may occur via the Na/Ca exchanger, the normal efflux pathway, and not the Ca-uniporter, the normal influx route. If this is the case, then depletion of myocyte Na + should inhibit Ca 2 + uptake, and collapse of the mitochondrial membrane potential (Δψ m ) would inhibit the uniporter. To test these hypotheses, isolated rat myocytes were exposed to metabolic inhibition, to mimic hypoxia, and [Ca 2 + ] m and [Ca 2 + ] c determined by selective loading of indo-1 into these compartments. Δψ m was determined using rhodamine 123. Following metabolic inhibition, [Ca 2 + ] m was significantly lower in Na-depleted cells than controls (P<0.001), [Ca 2 + ] c was approximately the same in both groups, and mitochondria depolarised completely. Thus Na-depletion inhibited mitochondrial Ca 2 + uptake, suggesting that Ca 2 + entry occurred via Na/Ca exchange, and the collapse of Δψ m during metabolic inhibition is consistent with inactivity of the Ca-uniporter.