99mTc-labeled 1-thio-beta-D-glucose (99mTc-1-TG) was synthesized under different ligand concentrations. 1-Thio-beta-D-glucose (1-TG) showed different chemical properties and higher stability at acid pH as compared to neutral and basic pH. Under different ligand concentrations, 99mTc-1-TG was obtained with high labeling efficiency of >97%, but the HPLC chromatogram varied with the ligand concentration. The results of a tumor cell uptake assay also depended on the ligand concentration. With the lowest ligand concentration tested (0.5 mg/ml of 1-TG), we obtained the highest uptake. With different glucose and insulin concentrations, 99mTc-1-TG uptake was not significantly different from that of [18F]FDG.