Background: Perioperative myocardial infarction adversely affects survival after cardiac operations. In animal studies Na+/H+ exchange inhibitors protect hearts against ischemia-reperfusion injury, but human trials have failed to consistently show beneficial effects. The aim of this study was to investigate whether the combination of the Na+/H+ exchange inhibitor cariporide and the β-blocker metoprolol additively protects hearts from severe ischemia-reperfusion injury. Methods: Isolated Langendorff-perfused rat hearts were randomly assigned to a vehicle-treated control group, or groups with 10-minute preischemia infusions of cariporide (10 μmol/L), metoprolol (10 μmol/L), or both cariporide and metoprolol. The hearts were then subjected to 20 minutes of global ischemia followed by 60 minutes of reperfusion. At the end of reperfusion, the hearts were randomly assigned to undergo either infarct size measurements or left ventricular mitochondrial function analyses. Results: The combination of cariporide and metoprolol limited infarct size significantly compared with control group or cariporide alone (5% ± 1% versus 58% ± 9% or 38.4% ± 4% of risk zone; p < 0.05). Cariporide alone did not reduce infarct size significantly as compared with the control group. As compared with the control group, cariporide and metoprolol decreased mitochondrial calcium content (6.4 ± 1.2 versus 10.2 ± 1.1 nmol/mg protein; p < 0.05), and increased respiratory control ratio (9.5 ± 0.6 versus 5.3 ± 0.7; p < 0.05). However, hearts treated with cariporide or metoprolol alone did not show significant improvement in mitochondrial calcium content (7.8 ± 1.2 and 7.8 ± 1.5 nmol/mg protein) or respiratory control ratio (5.0 ± 0.7 and 7.3 ± 0.7). Conclusions: The combination of cariporide and metoprolol additively limits infarct size after severe ischemia-reperfusion injury in an isolated rat heart model. Infarct size reduction occurs in association with protection from increased mitochondrial calcium content after reperfusion. © 2007 The Society of Thoracic Surgeons.