AIM: To explore and compare the radiochemical behavior and biological property of anti-sense oligonucleotide (ASON) labeled with technetium-99m using N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycl ine (NHS-MAG3) and hydrazinonictinamide derivative (HYNIC).METHODS: After HYNIC and NHS-MAG3 were synthesized, ASON was labeled with technetium-99m using HYNIC and NHS-MAG3 as a bifunctional chelator.The in vivo and in vitro stability, binding rates of labeled compounds to serum albumen, biodistribution of 99mTcMAG3-ASON and 99mTc-HYNIC-ASON in BALB/C mouse and its HT29 tumor cellular uptake were compared.RESULTS: The labeling efficiency and stability of 99mTcMAG3-ASON were significantly higher than those of 99mTcHYNIC-ASON (P = 0.02, and P = 0.03, respectively). 99mTcNAG3-ASON had a significantly lower rate of binding to serum albumen than 99mTc-HYNIC-ASON (P ＜ 0.05). In contrast to 99mTc-HYNIC-ASON, the biodistribution of 99mTcMAG3-ASON was significantly lower in blood, heart, liver and stomach (P ＜ 0.05), slightly lower in intestines and spleen (P ＞ 0.05) and significantly higher in lung and kidney (P ＜ 0.05). The HT29 tumor cellular uptake rate of 99mTcMAG3-ASON was significantly higher than that of 99mTcHYNIC-ASON (P ＜ 0.05).CONCLUSION: 99mTc-MAG3-ASON shows superior radiochemical behaviors and biological properties than 99mTc-HYNIC-ASON. 99mTc-MAG3-ASON is a potential radiopharmaceutical agent for in vivo application.