Intracellular calcium overload has been implicated in postischemic reperfusion injury. In myocytes, mitochondrial free calcium concentration ([Ca2+]m), not cytosolic free calcium concentration ([Ca2+]c), overload is related to reoxygenation injury. We tested the hypothesis that [Ca2+]m, not [Ca2+]c, overload is an important mediator of reperfusion injury in whole hearts. [Ca2+]m and [Ca2+]c were assessed using indo 1 fluorescence in isolated rat hearts subjected to 45 min of ischemia and 20 min of reperfusion. Ruthenium red (RR), a selective inhibitor of mitochondrial calcium uptake at 0.025 microM, attenuated the increase of [Ca2+]m (4% RR vs. 57% control) over preischemic levels (230 +/- 10 nM) but did not affect the increase of systolic [Ca2+]c (990 +/- 100 nM RR vs. 1,010 +/- 130 nM control). This was associated with improved recovery of left ventricular developed pressure (61% RR vs. 37% control) and attenuation of the increase of diastolic pressure (34 mmHg RR vs. 47 mmHg control). Contractile recovery was related to the degree of [Ca2+]m overload in both control and RR hearts (r2 = 0.47, P = 0.001). This study is the first to demonstrate that [Ca2+]m, and not [Ca2+]c, overload is related to reperfusion injury in intact beating hearts.