The study of flow dynamics on the coronary microcirculation of the intact beating heart has been greatly hampered by the thickness of the tissue and its continual movement. Using fluorescent microspheres and video-image processing, we have devised a means to measure an index of blood velocities. Measurements were made on open-chest anesthetized cats without any artificial restraints to the heart motion. However, advantages exist for minimizing mechanical motion, therefore, a heart holder based on a vacuum concept, along with modified versions of previously demonstrated methods, were tested. Results showed that the vacuum holder provided more restraint to epicardial motion than the pericardium alone, and despite large differences in epicardial motion between the two, overall flow measurements of the microspheres were comparable. Other methods of immobilizing the heart usually did not yield normal flow velocities. This video analysis method allows for the reproducible measurement of particle velocities at time points throughout the cardiac cycle. In keeping the normal heart dynamics intact, more realistic results from the study of coronary blood flows can be obtained.