Abstract: Objective To establish the radiolabeling method of exendin-4, an glucagon-like peptide 1 (GLP-1) receptor agonist, with ¹³¹I and observe the biodistribution of ¹³¹I-exend in-4 in normal mice. Methods Radiolabeling of exendin-4 with ¹³¹I was performed by chloramine-T method. The labeling ratio and radiochemical purity were determined using paper chromatography and TLC. The octanol-water partition coefficient and stability in vitro in saline and normal human serum of labeled polypeptide were also determined. The biodistribution of ¹³¹I-exendin-4 in normal mice were measured at 1, 3, 6, 12, 24 h after intravenous injection. SPECT was performed in 3 male mice after injection of ¹³¹I-exendin-4 to observe the dynamic distribution of the labeled exendin-4. Results The labeling yield of ¹³¹I-exendin-4 was more than 85%. The ¹³¹I-exendin-4 had good stability in saline and normal human serum. The octanol-water partiton coefficient lg P=-1.002. The biodistribution in mice showed that ¹³¹I-exendin-4 had rapid blood clearing, and the radioactivity in kidney was obviously higher than that of other organs as indicated by their uptake of (51.54±13.51)%ID/g at 1 h and (11.61±0.94)%ID/g at 12 h, respectively. Organs such as stomach, lung, intestine and bone had transient radioactive uptake, and then after 3 h the radioactivity in those organs rapidly decreased. The radioactivity in other organs always kept at a low level. Conclusion ¹³¹I-exendin-4 can be easily labeled with a high labeling ratio with stability in vitro. It has good distribution characteristics in vivo, and is cleared mainly by renal excretion due to its hydrosolubility.