Twenty minutes of ischemia in canine cardiac muscle produced a 50% to 60% inhibition of the mitochondrial ATPase. The inhibition has been shown to be triggered by a drop in cell pH under the non-energizing conditions which prevail in ischemic cells (Rouslin, W J Biol Chem 258, 9657-9661 (1983). In the present study we showed that the ATPase inhibition produced in situ in ischemic cardiac muscle was preserved in submitochondrial particles (SMP) prepared from mitochondria isolated from the ischemic tissue. The ischemic SMP ATPase was 45 +/- 3% as active as that of control particles. Measurements of the amounts of ATPase inhibitor protein of Pullman and Monroy present in extracts of control and ischemic SMP by two independent methods, titration of rat heart SMP ATPase and radioimmunoassay, revealed that control SMP contained 62 +/- 4% as much inhibitor as ischemic SMP as estimated by the titration procedure and 66 +/- 3% as much as estimated by the RIA. The results suggest that about one-third of the inhibitor was displaced from the control SMP. Finally, submitochondrial particles prepared from 20 min ischemic heart muscle showed a 2.5-fold increase in ATPase specific activity and a concomitant release of 35% of their inhibitor as a result of subsequent reenergization in vitro. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented both ATPase reactivation and inhibitor release. These findings support the hypothesis that the observed in situ ATPase inhibition is inhibitor protein mediated. Moreover, they suggest a pathophysiological function for the inhibitor protein in cardiac muscle.