The fluorescent microsphere method is one of the current techniques to determine regional blood flow in various organs. The purpose of this study was to examine the suitability of fluorescent microspheres for serial measurement of regional bone blood flow. Six anesthetized female New Zealand rabbits received five left ventricular injections of fluorescent microspheres in 20-minute intervals. To test the precision of the measurement two types of fluorescent microspheres were injected simultaneously at the first and last injections. Blood flow was calculated in the kidneys, lungs, brain, femurs, and tibias after measuring the fluorescence intensity in each reference blood and tissue sample. Comparison of blood-flow values obtained by simultaneously injected microspheres showed an excellent correlation and a minimal percentage difference at the first and last injections, indicating valid measurements of regional bone blood flow. No significant differences were observed when comparing blood flow in the corresponding regions of bones on the right side and left side. Mean blood flow in the femur and tibia significantly increased at the fourth injection whereas flow distribution within the femur and tibia essentially remained unchanged throughout the experiment. Comparison of blood flow values obtained by simultaneously injected microspheres showed moderate agreement for the kidneys and lungs at the last injections. Because this finding might be attributable to disturbances of microcirculation caused by accumulation of spheres in high-flow organs, the increase in regional bone blood flow observed in our experiments has to be interpreted carefully. This study showed that bone blood flow can be determined reliably in anesthetized rabbits by as many as three serial injections of fluorescent microspheres.