We have developed Tc-99m labeled activated carbon microspheres (99mTc-CH44) for use in mammary lymphoscintigraphy and detailed analysis of mammary lymph flow. 99mTc-CH44 was prepared according to the EDTA complex method. The labeling rate immediately after preparation was 97.7 +/- 2.6% at 37 degrees C and was 94.9 +/- 2.6% after 24-hour continuous shaking. The release of 99mTcO4- from 99mTc-CH44 was determined in vitro, using a simulation model. In this assay, the 99mTcO4- release rate in one hour was 6.6% and that in 6 hours was 38%. When 2ml (2mCi, 74MBq) of 99mTc-CH44 was injected into the mammary glands of 12 patients with breast tumors, the axillary, subclavian and parasternal lymph nodes of 10 patients were visualized 60 minutes later. In analysis of lymph flow in 25 rats using 99mTc-CH44 the lymph flow from the foot pad through the popliteal to the intraperitoneal lymph nodes was known to have only one route when examined by gross finding and histology. However, the examination using RI-technique indicated two routes. The examination in 9 patients with breast cancer indicated that the use of 99mTc-CH44 allowed detailed analysis of lymph flow. These results suggest that 99mTc-CH44 is useful for the preoperative mammary lymphoscintigraphy and detailed analysis of mammary lymph flow.