Identification of myocardial infarction (MI) by imaging is critical for clinical management of ischemic heart disease. Iodine-123-labeled hypericin (123I-Hyp) is a new potent infarct avid agent. We sought to compare target selectivity and organ distribution between 123I-Hyp and the myocardial perfusion agent, technetium-99m-labeled hexakis [2-methoxy isobutyl isonitrile] (99mTc-Sestamibi) in rabbits with acute MI. Hypericin was radiolabeled with 123I using iodogen as oxidant, and 99mTc-Sestamibi was prepared from a commercial kit and radioactive sodium pertechnetate. Rabbits (n = 6) with 24-hour-old MI received 123I-Hyp intravenously and received 99mTc-Sestamibi 9 hours later. They were studied by dual-isotope simultaneous acquisition micro single photon emission computed tomography/computed tomography (DISA-μSPECT/CT), tissue gamma counting (TGC), autoradiography, and histology. After purification, 123I-Hyp was obtained with radiochemical purity around 99%. DISA-μSPECT/CT images showed 123I-Hyp retention in infarcted but not in normal myocardium. By TGC, accumulation values reached 1.175 ± 0.096 percentage of injected dose per gram (%ID/g) and 0.028 ± 0.007%ID/g in infarcted myocardium and normal myocardium with high tracer concentration in liver, intestines, and gallbladder. 99mTc-Sestamibi was prepared with radiochemical purity over 95%. DISA-μSPECT/CT showed no accumulation in MI and high initial radioactivity levels in normal myocardium that were rapidly cleared as confirmed by TGC (0.011 ± 0.003%ID/g). Liver and intestines were clearly visualized. By TGC, gallbladder and kidneys show moderate 99mTc-Sestamibi uptake. The selectivity of 123I-Hyp for infarcted myocardium and 99mTc-Sestamibi for normal myocardium was confirmed. 123I-Hyp distribution in rabbits is characterized by hepatobiliary excretion. 99mTc-Sestamibi undergoes hepatorenal elimination.