Radioactive microspheres have long been in use to measure blood flow distribution to various vascular beds. Their drawbacks are the short half-lives of the radioactive material, the need for appropriate care in handling and disposing of such material, and their relative expensiveness. We investigated the use of fluorescent microspheres as indicators of coronary blood flow distribution in a canine model. Radioactive (125I) microspheres were used as a comparison standard. Four colors of fluorescent microspheres were used: blue, yellow-green, orange, and red, having emission frequencies ranging from 385 to 605 nM. The experiments were carried out in dogs under pentobarbital anesthesia, in which the microspheres were given during pump perfusion of the left circumflex artery with the animal's own blood. The hearts were removed, fixed for 3 days in 10% formalin, and sectioned. Samples from the endocardial, myocardial, and epicardial layers were read on a gamma counter. The fluorescent microspheres were extracted from the same tissues into ethyl acetate, and read in a fluorescence spectrophotometer at the appropriate excitation/emission frequencies. Comparable results were obtained from the two methods, with good sensitivity and resolution of dye colors, using the fluorescent microspheres.