Matrix metalloproteinases (MMP) are a family of enzymes that can degrade myocardial proteins. Increases in MMP activity occur in the setting of myocardial ischemia/reperfusion (I/R) injury. The role that MMP activation may play in degrading structural components of the myocardium is not fully known. Furthermore, the role that specific MMPs play in compromising myocardial structure/function during I/R injury is also not well understood. To address these issues we utilized an ex vivo rat heart model of I/R injury. For this purpose novel zinc-binding group (ZBG), semi-selective and well characterized broad-spectrum MMP inhibitors (MMPI), as well as minocycline, were used. Hearts were subjected to 20-30 min of global no-flow ischemia and 30 min reperfusion while heart rate and chamber pressures were recorded. Markers for damage to muscle (i.e. troponin I and creatine kinase in coronary effluent), vascular integrity (coronary flow rate, end diastolic pressure, heart weight) and extracellular matrix damage (collagen fragments) were measured. Control (vehicle treated) I/R groups were generated. Vehicle/compounds were administered during the period of stabilization and until the end of the study. The use of the novel ZBG thiomaltol, semi-selective MMPI (ssMMPI) PY-2, 1,2-HOPO-2 and gelatinase inhibitor I targeting mostly MMP-2, -3, -8, -12 and -13, as well as the pleiotropic agent minocycline which can also inhibit MMPs, led to significant time/dose dependent increases in the recovery of contractile function (measured as heart rate-pressure product). Coronary flow during reperfusion was significantly improved by the use of the ssMMPIs and minocycline. PY-2, 1,2-HOPO-2 and minocycline significantly reduced troponin I release. Global MMP activity in coronary effluent was significantly reduced only by 1,2-HOPO-2. Reduced levels of tissue edema and collagen degradation were observed with 1,2-HOPO-2. The use of the broad-spectrum MMPI (bsMMPI) CGS27023A or PD166793 marginally improved contractile function vs. controls. ELISA vs. MMP-2, -3, -13 were used to detect MMPs in perfusate documenting the presence of MMP-2 during reperfusion. We can conclude that ssMMPIs and minocycline can protect isolated hearts from I/R injury. MMP-2 is likely involved in mediating injury. Data suggests protection of the vascular compartment by MMPI may be critical during cardiac I/R injury.