For the investigation of myocardial perfusion and left ventricular pump function, advanced radioisotopic techniques have been established. New developments in radiopharmacology and single-photon emission computed tomography have recently enabled the investigation of parameters of regional energy metabolism in well defined areas of the heart muscle. For this purpose, various iodine (123I)-labeled free fatty acids (FFA) have been synthesized. The diagnostic application of labeled FFA in heart disease may be important, since FFA are the preferred substrates for cardiac energy production at rest in the fasting state. In addition, regional myocardial FFA uptake and regional myocardial blood flow are tightly coupled in normal myocardium with beta-oxidation which is extremely sensitive to oxygen deprivation. This article outlines the basic physiologic pathways of FFA in normal and ischemic myocardium and reviews the results of animal experiments validating the application of these principles for metabolic imaging of the heart by means of the aromatic radioiodinated FFA, 15-(p-iodophenyl)pentadecanoic acid. In addition, the development, physiologic properties, and potential applications of a new generation of 3-methyl-substituted radioiodinated fatty acids that show high myocardial uptake but prolonged retention are discussed.