We have used mitochondrial entrapment of 2-deoxy-D-[3H]glucose (2-DG) to demonstrate that recovery of Langendorff-perfused rat hearts from ischemia is accompanied by reversal of the mitochondrial permeability transition (MPT). In hearts loaded with 2-DG before 40 min of ischemia and 25 min of reperfusion, 2-DG entrapment [expressed as 10(5) x (mitochondrial 2-[3H]DG dpm per unit citrate synthase)/(total heart 2-[3H]DG dpm/g wet wt)] increased from 11.1 +/- 1.3 (no ischemia, n = 4) to 32.5 +/- 1.9 (n = 6; P < 0.001). In other experiments, 2-DG was loaded after 25 min of reperfusion to determine whether some mitochondria that had undergone the MPT during the initial phase of reperfusion subsequently "resealed" and thus no longer took up 2-DG. The reduction of 2-DG entrapment to 20. 6 +/- 2.4 units (n = 5) confirmed that this was the case. Pyruvate (10 mM) in the perfusion medium increased recovery of left ventricular developed pressure from 57.2 +/- 10.3 to 98.9 +/- 10.8% (n = 6; P < 0.05) and reduced entrapment of 2-DG loaded preischemically and postischemically to 23.5 +/- 1.5 (n = 4; P < 0. 001) and 10.5 +/- 0.5 (n = 4; P < 0.01) units, respectively. The presence of pyruvate increased tissue lactate content at the end of ischemia and decreased the effluent pH during the initial phase of reperfusion concomitant with an increase in lactate output. We suggest that pyruvate may inhibit the MPT by decreasing pHi and scavenging free radicals, thus protecting hearts from reperfusion injury.