Abstract: The charge is an important characteristic of a drug molecule. The overall charge on a drug affects its pharmacological properties in vivo. In this study, (S)-methyl 3-(4-hydroxyphenyl)-2-(3-(2-nitro-1H-imidazol-1-yl)propanamido)propanoate (NI-Y-M), (S)-3-(4-hydroxyphenyl)-2-(3-(2-nitro-1H-imidazol-1-yl)propanamido) propanoic acid (NI-Y) and (S)-N-(3-aminopropyl)-3-(4-hydroxyphenyl)-2-(3-(2-nitro-1H-imidazol-1-yl)propanamido)propanamide (NI-Y-A) were synthesized and radioiodinated with ¹³¹I. All the radioiodinated compounds are obtained in >95% yield determined by HPLC and stable at room temperature for 24 h in vitro. The paper electrophoresis results indicate that the compounds formed are different in charge,［¹³¹I］NI-Y-M, ［¹³¹I］NI-Y, and ［¹³¹I］NI-Y-A are neutral, negative and positive, respectively. The biodistribution results in mice bearing S180 tumor demonstrate that all of the ［¹³¹I］NI-Y-M, ［¹³¹I］NI-Y and ［¹³¹I］NI-Y-A accumulate in tumor fast and slowly clear from it. The uptakes in tumor are higher than those in muscle, heart, brain and spleen. The tumor-to-tissue ratios increase along with time. At 4 h post injection, the uptake ratios of tumor-to-muscle and tumor-to-blood indicate that among them the positively charged ［¹³¹I］NI-Y-A is the best hypoxia agent.