Effects of hyaluronidase on myocardial water content and distribution, and on coronary vascular hemodynamics and endothelial cell transport function were assessed in isolated rabbit hearts during 3.5 hours of reperfusion after 30 minutes of global, no-flow ischemia. In nonischemic control hearts, perfusion pressure, left ventricular end-diastolic pressure, maximum +dP/dt, and intravascular clearance of radiolabeled albumin remained constant during 5 hours of continuous perfusion, while the mean-transit time and vascular into extravascular space clearance of radiolabeled albumin increased 1.5x and 2.5x baseline, respectively. During reperfusion after 30 minutes of no flow, perfusion pressure increased 53% and interstitial fluid volume increased 2-fold, while left ventricular end-diastolic pressure and maximum +dP/dt returned to control levels. The rate of intravascular clearance of radiolabeled albumin decreased 38%, and the mean-transit time and vascular-into-extravascular space clearance of albumin increased ~3x and 5x baseline, respectively. Hyaluronidase blocked the ischemia-reperfusion-induced increases in total water content and in interstitial fluid volume and reduced the increases in perfusion pressure and mean-transit time of radiolabeled albumin by 40% and 45%, respectively, but did not prevent the increase in albumin vascular-into-extravascular space clearance and the decrease in albumin clearance from the coronary vasculature. These findings indicate that hyaluronidase does not prevent ischemia-reperfusion-induced increases in albumin permeation of the coronary vasculature, and suggest that its protective effect on ischemic myocardium is mediated, instead, by reducing interstitial edema and vascular resistance.