Right atrial, pulmonary artery, pulmonary capillary, pulmonary artery wedge, and systemic blood pressures of strenuously exercising horses increase markedly. As a consequence, myocardial metabolic O2 demand in exercising horses must be high. Experiments were, therefore, carried out on 9 healthy, exercise-conditioned horses (2.5 to 8 years old; 481 +/- 16 kg) to ascertain the regional distribution of myocardial blood supply in the atria and ventricles at rest and during exercise. Blood flow was measured, using 15-micrometer-diameter radionuclide-labeled microspheres that were injected into the left ventricle while reference blood samples were being withdrawn at a constant rate from the thoracic aorta. Myocardial blood flow was determined at rest and during 2 exercise bouts performed on a high-speed treadmill at 8 and 13 m/s (0% grade). The sequence of exercise bouts was randomized among horses, and a 60-minute rest period was permitted between exercise bouts. There was considerable heterogeneity in the distribution of myocardial perfusion in the atria and the ventricles at rest; the right atrial myocardium received significantly (P < 0.05) less perfusion than did the left atrium, and these values were significantly (P < 0.05) less than those for the respective ventricular myocardium. The right ventricular myocardial blood flow also was significantly less than that in the left ventricle. With exercise, myocardial blood flow in all regions increased progressively with increasing work intensity and marked coronary vasodilation was observed in all cardiac regions. During exercise at 8 or 13 m/s, right and left atrial myocardial blood flows (per unit weight basis) were not different from each other. Although at treadmill speed of 8 m/s, left ventricular myocardial blood flow exceeded that in the right ventricle, this was not the case at 13 m/s, when perfusion values (per unit weight basis) became similar.