Mitochondrial calcium overload has been suggested as a marker for irreversible injury in the ischemic heart. A new technique is used to measure dynamic changes in mitochondrial free calcium concentration ([Ca2+]m) in electrically stimulated (0.2 Hz) adult rat cardiac myocytes during exposure to anoxia and reoxygenation. Cells were incubated with indo-1 AM, which distributes in both the cytosol and mitochondria. After Mn2+quenching of the cytosolic signal, cells were exposed to anoxia, and the residual fluorescence was monitored. [Ca2+]. averaged 94±3 nM (n=16) at baseline, less than the baseline diastolic cytosolic free calcium concentration ([Ca2+]c, 124±4 nM,n=12), which was measured in cells loaded with the pentapotassium salt of indo-1. [Ca2+]mand [Ca2+]crose steadily only after the onset of ATP-depletion rigor contracture. At reoxygenation 35 minutes later, [Ca2+]cfell rapidly to preanoxic levels and then often showed a transient further rise. In contrast, [Ca2+]mshowed only a slight transient fall and a secondary rise at reoxygenation. At reoxygenation, cells immediately either recovered, demonstrating partial relengthening and retaining their rectangular shape and response to stimulation, or they hypercontracted to rounded dysfunctional forms. Recovery occurred only in cells in which [Ca2+]mor [Ca2+]cremained below 250 nM before reoxygenation. Early during reoxygenation, [Ca2+]mremained higher in cells that hypercontracted (305±36 nM) than in cells that recovered (138±9 nM,p<0.05), whereas [Ca2+]cdid not differ between the two groups (156±10 versus 128±10 nM, respectively; p=NS). The role of the sarcoplasmic reticulum in Ca2+regulation was evaluated in cells (n=16) pretreated with thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. During anoxia [Ca2+]cand [Ca2+]mrose as they did without thapsigargin pretreatment. At reoxygenation, the rapid fall in [Ca2+]cwas blunted, and [Ca2+]mshowed an immediate increase in these cells, demonstrating the importance of the sarcoplasmic reticulum in postanoxic Ca2+regulation. In summary, cellular hypercontracture is not associated with a sudden and massive rise in [Ca2+]cimmediately after reoxygenation. The basis for the relation between [Ca2+]mand cellular recovery as well as the mechanisms underlying the observed changes in [Ca2+]mremain to be defined.