Objective: Sarcoplasmic reticulum dysfunction may contribute to calcium (Ca 2 + ) overload during myocardial reperfusion. The aim of this study was to investigate its role in reperfusion injury. Methods: Open chest dogs undergoing 15 min of left anterior descending coronary artery occlusion and 3 h of reperfusion were randomized to intracoronary infusions of 0.9% saline, vehicle, or the Ca 2 + channel antagonist, nifedipine (50 μg/min from 2 minutes before to 5 minutes after reperfusion). After each experiment, transmural myocardial biopsies were removed from ischemic/reperfused and nonischemic myocardium in the beating state and analyzed for (i) sarcoplasmic reticulum protein content (Ca 2 + ATPase, phospholamban, and calsequestrin) by immunoblotting and (ii) Ca 2 + uptake by sarcoplasmic reticulum vesicles with and without 300 micromolar ryanodine or the Ca 2 + ATPase activator, antiphospholamban (2D12) antibody. Results: Contractile function did not recover in controls and vehicle-treated dogs after ischemia and reperfusion (mean systolic shortening, -2 ± 2%), but completely recovered in nifedipine-treated dogs (17 ± 2%, p = NS vs. baseline, p < 0.01 vs. control). Ventricular fibrillation occurred in 50% of controls and vehicle dogs and 0% of nifedipine-treated dogs (p < 0.01). Ca 2 + uptake by the sarcoplasmic reticulum vesicles was severely reduced in ischemic/reperfused myocardium of controls and vehicle dogs (p < 0.01 vs. nonischemic). Ryanodine and the 2D12 antibody improved, but did not reverse the low Ca 2 + uptake. Protein content was similar in ischemic/reperfused and nonischemic myocardium. In contrast, Ca 2 + uptake and the responses to ryanodine and 2D12 antibody were normal in ischemic/reperfused myocardium from nifedipine-treated dogs. Conclusion: Dysfunction of the sarcoplasmic reticulum Ca 2 + ATPase pump correlates with reperfusion injury. Reactivation of Ca 2 + channels at reperfusion contributed to Ca 2 + pump dysfunction. Ca 2 + pump injury may be a critical event in myocardial reperfusion injury.