Fluorescent microspheres have been used in multiple particle tracking studies. Our laboratory is working towards the use of fluorescent microsphere particle tracking in the study of the retinal circulation. The first stage of this project was to develop a technique for measurement and analysis of the particle tracking data. We intravenously injected 2.0 microm diameter, polystyrene microspheres, labeled with fluorescent dye, in anesthetized rats. Using a microscope with a fluorescence attachment, we viewed the fluorescent particles flowing through the surface vessels of the intestine. Data was recorded then analyzed frame by frame. The path of each microsphere was plotted on a transparency sheet and measured. Using a magnification ratio, the velocity and blood flow rate were calculated for 235 particles in five different blood vessels. We found this technique to be an effective method for studying blood flow in an arteriole. The results were comparable to other blood flow studies and showed characteristic trends of typical laminar blood flow.