Gold nanoparticles, have been found highly effective in cancer diagnosis and therapy, due to their own physicochemical properties, easy functionalization of their surface with different chemical entities, low toxicity and biocompatibility. On the other hand 99mTc is used due to the emission of low energy γ-rays (140keV), suitable half-life (6.02 h) and good availability from 99Mo/99mTc generators. The aim of the study, is to radiolabel Gold Nanoparticles (AuNPs) of different sizes with thiol ligands (as an anchor for AuNPs surface) that allow direct labeling with 99mTc-carbonyls. Thiols are the most important type of stabilizing molecules for AuNPs of any size because thiols lead to the formation of strong Au-S bonds. After functionalization and radiolabeling of the AuNPs, we perform in vitro stability studies in human serum as well as in cysteine and histidine solutions, in order to evaluate their stability in vivo. Furthermore, cytotoxicity studies to invest the potential toxic effect of the formed complexes, as well as biodistribution studies in tumor-bearing animal models, are carried out.