To evaluate morphologic changes and myoglobin content in normal, ischemic and necrotic myocardium, the authors studied human (n = 13) and dog (n = 28) myocardium by triphenyltetrazolium chloride staining, light and electron microscopy, periodic acid-Schiff stain for glycogen loss, and by an immunoperoxidase technique. Myocardium from autopsied patients with infarction 10-24 hours old showed loss of myoglobin from necrotic fibers. Dogs with infarcts after 3 hours or more of coronary occlusion showed myoglobin loss in fibers shown to be necrotic. In 4 dogs with 50% reduction in left main coronary artery flow for 3 hours, which demonstrated ischemia without necrosis (glycogen loss with no triphenyl tetrazolium chloride evidence of necrosis), myoglobin staining in myocardial sections was similar to nonischemic and positive control tissues. By comparison of immunoperoxidase staining with concomitant study by light and electron microscopy and histochemistry, loss of myoglobin from necrotic myocardium was demonstrated, while ischemic but not necrotic fibers stained normally. These findings indicate that necrosis is necessary for myoglobin loss from myocardium to be detected by this immunoperoxidase technique.