Heart attacks result from reduced or blocked blood flow through major coronary arteries, resulting in permanent damage to heart muscle. Coronary blood flow (CBF) is thus important to measure in experimental animal models of heart disease. A standard method to measure CBF uses tracer microspheres (0 = 15 µm) injected into the left ventricle that flow through coronary arteries but cannot pass through capillaries and so become trapped in heart muscle. Previously, radioactive or colored microspheres have quantified the number of tracers trapped in the muscle. Fluorescent microspheres offer a more recent and more sensitive measurement mode. However, fluorescent microspheres have not often been used to measure CBF in small animals (rats, mice) that are now the most common animal models used in heart research. This thesis aimed to develop the techniques for use of fluorescent microspheres to measure CBF in rat animal models used by the cell biology laboratories at UMDNJ-Newark. Two non-overlapping fluorescent wavelengths were chosen (yellow-green; red). Using a spectrophotometer, fluorescence intensity was calibrated for known numbers of microspheres (set via controlled dilution). CBF in two rats was measured at rest and during maximal vasodilation (adenosine) using procedures for colored microspheres. After euthanasia, hearts were removed, and blood samples and left ventricular tissue were processed using a sedimentation method for full recovery of fluorescent microspheres, which were scanned through the spectrophotometer to count fluorescence intensity. Using the predetermined calibration curve, the number of microspheres in each sample was determined; from this CBF was calculated. CBF averaged 5.9 ml/min/g at rest, which was within the normal range for rats quoted in recent literature. With maximal vasodilation, CBF increased to an average of 12.9 ml/min/g, which indicated a coronary flow reserve that was 2.2 times the resting level. The same value for coronary flow velocity reserve (2.2) was measured in 6 rats using Doppler echocardiography. The consistency of these results suggests that the procedures developed for fluorescent microspheres lead to repeatable and reliable measurement of coronary blood flow in rats.