The effects of intravenous infusions of isoproterenol (0.1, 1.0 or 2.5 micrograms/kg/min for 1 hr) on the transmural distribution of myocardial high-energy phosphates and glycolytic intermediates were determined in anesthetized, open chested dogs. The transmural distribution of blood flow across the left ventricular wall was determined after serial injections of 15 mu radioactively labeled microspheres. A biopsy of the posterolateral wall was frozen in liquid nitrogen, divided into epicardial, midmyocardial and endocardial thirds, and assayed for metabolites. The remainder of the heart was processed for light and electron microscopy. At the infusion rate of 2.5 micrograms/kg/min, isoproterenol caused nonuniform reductions in ATP, phosphocreatine, total adenine nucleotides and glycogen, which were particularly depleted in the endocardium. Isoproterenol also caused nonuniform increases above baseline in blood flow (epicardial, 492%; midmyocardial, 197%; and endocardial, 131%). The endocardial/epicardial blood flow ratios, however, were reduced. Evidence of cellular damage was demonstrated by the presence of numerous contraction band lesions along with mitochondrial swelling and the appearance of electron-dense deposits. It is concluded that necrogenic infusions of isoproterenol impair myocardial energy production in all myocardial layers despite an overall increase in blood flow. There appears to be a link between the gradients in metabolites and the distribution of blood flow such that the endocardium becomes more vulnerable to injury.