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+]m 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+]m and [Ca2+]c rose steadily only after the onset of ATP-depletion rigor contracture. At reoxygenation 35 minutes later, [Ca2+]c fell rapidly to preanoxic levels and then often showed a transient further rise. In contrast, [Ca2+]m showed 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+]m or [Ca2+]c remained below 250 nM before reoxygenation. Early during reoxygenation, [Ca2+]m remained higher in cells that hypercontracted (305 +/- 36 nM) than in cells that recovered (138 +/- 9 nM, p less than 0.05), whereas [Ca2+]c did not differ between the two groups (156 +/- 10 versus 128 +/- 10 nM, respectively; p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)