The sentinel lymph node (SLN) is defined as the first regional lymph node to receive lymphatic drainage from a malignant tumor. Therefore, this node is a "sentinel" for second metastatic lymph node stations and for labeling regional tumor spread. For SLN detection, many surgeons preferred a combination of a preoperative injection of radiolabeled colloid and the intraoperative injection of blue dye. Under this combination protocol, nodes are considered to be "sentinel nodes" if they are radioactive and, blue. The aim of this study is to develop a new single agent that combines both detection methods. For this purpose Isosulfan Blue (ISB) was radiolabeled by Tc-99m with high labeling yield and stability. In vivo gamma scintigraphy studies were performed with rats. According to the scintigraphic studies, Tc-99m-ISB shows rapid and high accumulation in both axillary (ALN) and popliteal lymph node (PLN). After the imaging study, extremity was opened and nodes were scanned for the radioactivity. According to performed study the lymph nodes were clearly seen to become blue and carried compound was sufficient to allow identification with a gamma probe. In conclusion, Tc-99m-ISB has the potential to facilitate lymphatic mapping and subsequent sentinel node biopsy for solid malignancies such as breast cancer and melanoma.