99mTc-labeled mannosyl-neoglycoalbumin (NMA) was prepared and evaluated as a radiopharmaceutical for sentinel lymph node (SLN) identification, since 99mTc-labeled human serum albumin (HSA) rapidly cleared from injection sites. NMA was conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) and reacted with [99mTc](tricine)2 to prepare [99mTc](HYNIC-NMA)(tricine)2. After subcutaneous injection of [99mTc](HYNIC-NMA)(tricine)2 from murine foot pad, radioactivity levels in the popliteal and lumbar lymph nodes, the injection site and other tissues were compared with those of [99mTc](HYNIC-HSA)(tricine)2 and 99mTc-labeled colloidal rhenium sulfate ([99mTc]colloid). [99mTc](HYNIC-NMA)(tricine)2 demonstrated significantly higher radioactivity levels in the popliteal lymph node, the SLN in this model, than did [99mTc](HYNIC-HSA)(tricine)2 and [99mTc]colloid at 0.5, 1, and 6 h post-injection. [99mTc](HYNIC-NMA)(tricine)2 showed a dose-dependent decrease in the popliteal accumulation while the radioactivity levels in the blood, liver and spleen increased with an increase in the molar dose of NMA. [99mTc]colloid registered a decrease in the radioactivity levels in the popliteal lymph node, blood, liver, and spleen with dilution. However, the radioactivity levels at the injection site increased with dilution of [99mTc] colloid. Both [99mTc](HYNIC-NMA)(tricine)2 and [99mTc](HYNIC-HSA)(tricine)2 showed the radioactivity levels at the injection site similar each other. These findings indicated that an addition of a macrophage binding function to 99mTc-labeled HSA provided high and selective accumulation of the radioactivity in the SLN without affecting the elimination rate from the injection site. Such characteristics render [99mTc](HYNIC-NMA)(tricine)2 attractive as a radiopharmaceutical for SLN identification. This study also demonstrated that the number of non-radiolabeled colloidal particles and the molar dose of mannosylated compounds play a crucial role in the SLN accumulation.