Fluorescent microspheres (FM) were used in vivo to visualize and quantify an ischemic risk zone after experimental coronary artery occlusion (CAO). Anesthetized rats were subjected to CAO. After 5 min, FM were injected into the left ventricular cavity of seven rats. Six control rats received no FM. When examined with magnification under ultraviolet light, heart slices with FM showed a white-green particulate fluorescence in the nonischemic septum and a well-delineated transmural zone of nonfluorescence in the ischemic free wall. In hearts without FM, this fluorescence was not apparent nor was any perfusion defect observed. In other rats, relative regional myocardial blood flow in the nonfluorescent ischemic zone outlined by FM was measured using plastic radioactive microspheres and found to be approximately 12% of regional flow in normal areas where fluorescence was present. A close correlation (r = 0.92) existed between the amount of ischemic myocardium at risk as outlined by FM and that of necrotic myocardium as defined by tetrazolium staining after both 6 and 24 h of coronary occlusion. We conclude that FM is a promising technique in a small-animal model in which an ischemic risk zone can be established shortly after CAO and can be preserved for later quantification and analysis.