If the localization of musculoskeletal sarcomas could be visualized during surgery, it would be possible to completely resect the tumor with minimum damage to normal tissues and the patients could retain a functional limb. Therefore, we conducted the present study to clarify the usefulness of acridine orange (AO) for fluorovisualization of tumors using a mouse osteosarcoma model. At 2 hours after injection of 10 mg/kg AO to mice inoculated with MOS mouse osteosarcoma cells, fluorovisualization of mouse osteosarcoma reached the maximum level. Even a 1-mm-diameter lesion of pulmonary metastasis was visualized. The results suggested that AO may be useful for specific fluorovisualization of human osteosarcomas during surgery.