The fluorescent microsphere (FM) method is considered a reliable technique to determine regional bone blood flow (RBBF) in acute experiments. In this study, we verified the accuracy and validity of this technique for measurement of RBBF in a long-term experiment and examined RBBF after meniscectomy. Twenty-four anesthetized female New Zealand white rabbits (3 groups, each n = 8) received consecutive left ventricular injections of FM in defined time intervals after meniscectomy: group 1 from preoperation to 3 wk postoperation; group 2 from 3 to 7 wk postoperation; and group 3 from 7 to 11 wk postoperation. To test the precision of the FM method, two FM species were injected simultaneously at the first and last measurement. After the experiment, humeri, femora, tibiae, and reference organs (kidney, lung, brain) were removed and dissected according to standardized protocols. Fluorescence was determined in each reference blood and tissue sample, and blood flow values were calculated. Blood flow in kidney, lung, and brain revealed no significant difference between right and left side and remained unchanged during the observation period, thus excluding errors due to shunting and dislodging of spheres in our experiments. Comparison of relative bone blood flow values obtained by simultaneously injected FM showed an excellent correlation at the first and last injection, indicating valid RBBF measurements in long-term experiments. We found a significant increase in RBBF 3 wk after meniscectomy in the right tibial condyles compared with the nonoperated left side. Similar changes were found in the femoral condyles. RBBF in other regions of tibia, femur, and humerus revealed no significant differences between right- and left-sided bone samples of the same region. Our results demonstrate that the FM method is valid for measuring RBBF in long-term experiments. In addition, we were able to demonstrate that meniscectomy leads to an increase in RBBF in the tibial condyles at a very early stage. This increase might be caused by stress-induced alterations of the subchondral bone.