Determination of blood flow in tissues at risk for infarction is necessary for in vivo studies of the pathologic effects of vascular occlusion. Such blood flow measurements are traditionally carried out using radioactive microspheres. These are expensive and may pose a significant hazard to laboratory personnel. In addition, disposal of radioactive wastes, especially radioactive carcasses, is an increasingly expensive and inconvenient process. To avoid these problems, we have developed a modified procedure using fluorescent microspheres. To measure blood flow, spheres are injected intravenously and a blood sample is taken. Following cardiectomy, appropriate segments of left ventricular myocardium are digested with proteases to release the spheres, which are then purified through sucrose gradients. The spheres may be counted manually using an epifluorescence microscope, or by flow cytometry. Comparison of this method with the traditional radioactive microsphere procedure reveals similar results for the two methods. Fluorescent microspheres should provide a useful and accurate alternative method for measuring blood flow in studies of vascular disease.