Myocardial ischemia-reperfusion (I/R) is associated with the activation of matrix metalloproteinases (MMPs) and serine proteases. We hypothesized that activation of MMPs and the serine protease plasmin contribute to early cardiac myocyte death following I/R and that broad-spectrum protease inhibition with doxycycline (DOX) preserves myocyte viability. Rats treated daily with or without DOX beginning 48 h prior to experimentation were subjected to 30 min of coronary occlusion and 2 days of reperfusion. DOX pre-treatment reduced infarct size by 37%. DOX attenuated increases in MMP-9 and plasmin levels as determined by gelatin zymography and immunoblot, respectively. Neutrophil extravasation was unaltered by DOX as assessed by myeloperoxidase (MPO) activity. To examine the contribution of MMP-9 and plasmin to myocyte injury, cultures of neonatal rat ventricular myocytes (NRVMs) were treated for 48 h with 83 kDa MMP-9 or plasminogen in the presence or absence of DOX. MMP-9 treatment did not affect myocyte viability. Plasminogen treatment led to increased plasmin activity, resulting in loss of beta1-integrin, NRVM detachment and apoptosis. DOX co-treatment inhibited plasmin activity and preserved NRVM attachment, whereas co-treatment with the broad-spectrum MMP inhibitor GM6001 had no effect. These results indicate that plasmin causes disruption of myocyte attachment and viability independently of MMP activation in vitro and that inhibition of plasmin by DOX may reduce I/R-induced myocyte death in vivo through the inhibition of plasmin.