Objective: Mitochondrial [Ca²⁺] ([Ca²⁺]_m) rises in parallel with cytosolic [Ca²⁺] ([Ca²⁺]_c) following ATP-depletion rigor contracture induced by hypoxia in isolated cardiomyocytes. We investigated the pathways involved in the hypoxia induced changes in [Ca²⁺]_m by using known inhibitors of mitochondrial Ca²⁺ transport, namely ruthenium red, an inhibitor of the Ca²⁺ uniporter (the normal influx route) and clonazepam, an inhibitor of Na⁺/Ca²⁺ exchange, (the normal efflux route). Methods: [Ca²⁺]_m was determined from indo-1/am loaded rat myocytes where the cytosolic fluorescence signal had been quenched by superfusion with Mn²⁺. [Ca²⁺]_c was measured by loading myocytes with indo-1 pentapotassium salt during the isolation procedure. Cells were placed in a specially developed chamber for induction of hypoxia and reoxygenated 40 min after rigor development. Results: 50% of control cells hypercontracted upon reoxygenation; this correlated with a [Ca²⁺]_m or [Ca²⁺]_c higher than approximately 350 nM at the end of rigor. Clonazepam completely abolished the rigor-induced rise in [Ca²⁺]_m but not [Ca²⁺]_c. On reoxygenation [Ca²⁺]_m increased over the first 5 min and remained elevated whereas [Ca²⁺]_c fell. In the presence of ruthenium red a dramatic increase in [Ca²⁺]_m occurred 5–10 min after rigor development (the indo-1 fluorescence signal was saturated); [Ca²⁺]_c also increased but to a lesser extent. On reoxygenation, [Ca²⁺]_m fell rapidly even though cells hypercontracted and [Ca²⁺]_c remained elevated. Conclusions: During hypoxia following rigor development Ca²⁺ uptake into mitochondria occurs largely via the Na⁺/Ca²⁺ exchanger rather than the Ca²⁺ uniporter whereas on reoxygenation the transporters resume their normal directionality.