The effects of increased dietary cholesterol content 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 control hearts from rabbits fed normal chow, perfusion pressure, left ventricular end-diastolic pressure, maximum + dP/dt, and the rate of intravascular clearance of radiolabelled albumin remained constant during 5 hours of continuous perfusion, while the mean transit time of radiolabelled albumin increased 1.6 ± baseline. In ischemic hearts from rabbits fed normal chow, perfusion pressure increased 59% during reperfusion while left ventricular end-diastolic pressure and maximum +dP/dt returned toward control levels. The rate of intravascular clearance of radiolabelled albumin decreased 36%, and the mean transit time of albumin increased ∼3 ± baseline. Ischemia-reperfusion injury to the cardiac vasculature and musculature was markedly increased in hearts of rabbits fed chow supplemented with 2% cholesterol for 2–3 weeks compared to rabbits fed the same diet for a longer duration (5–16 weeks) or rabbits fed normal chow. Prior to ischemia, permeation of the coronary vasculature by albumin was increased twofold in rabbits fed cholesterol for 2–3 weeks while myocyte contractile function was normal relative to chow-fed controls or the group fed cholesterol for 5–16 weeks. These effects of acute cholesterol feeding precede occlusive atherosclerotic coronary artery disease and occur at plasma cholesterol concentrations one third of those in rabbits fed cholesterol for the longer duration. These findings suggest that the altered metabolic milieu associated with an abrupt increase in cholesterol consumption and/or a rapidly increasing plasma cholesterol concentration impairs the functional integrity of the coronary vasculature and leads to increased susceptibility of the heart to ischemia-reperfusion injury.