A method for labeling polypeptide (insulin) with technetium- 99(99mTc) was established without marked loss of biological activity. Following reduction of intrinsic disulfide bonds by mercaptoethanol and purification on a Sephadex G50 column, the polypeptide was labeled with 99mTc by transchelation from methylene diphosphonate (MDP). 99mTc labeled insulin was identified by thin layer chromatograph (TLC) and the change of blood sugar of mice injected, their hypoglycemic shock symptom was also observed. Six hours after labeling, the dissociation of labeled insulin was only 3%, Mom then on to 24 h, there was no more dissociation. The blood sugar concentration of mice injected with the mercaptoethanol-reduced insulin was (5.0±3.2)μmol.L-1, while those injected with the original insulin was (1.4±1.2)μmol.L-1, the difference was significant (Q test, p <0.01). Blood sugar concentration of the mice was 0.3±0.2μmol.L-1 for the labeled insulin, and was about the same with that for the original insulin. The labeling efficiency was 74.31% for the labeled insulin, whereas the original insulin cannot be labeled with 99mTc. The result suggests that while disulfide bonds of polypeptide were reduced by mercaptoethanol, it became free sulfhydryl group, and its bioactivity descended. Then free sulfhydryl group was chelated with 99mTc under mild condition, restablishing the disulfide bond, therefore, the bioactivity came back. The 99mTc-labeled insulin was stable during 24h. A reduction of intrinsic disulfide bonds by mercaptoethanol and purification on a Sephadex G50 column, the polypeptide was labeled with 99mTc by transchelation from methylene diphosphonate (MDP). 99mTc labeled insulin was identified by thin layer chromatography (TLC) and the change of blood sugar of mice injected, their hypoglycemic shock symptom was also observed. Six hours after labeling, the dissociation of labeled insulin was only 3% Mom then on to 24 h, there was no more dissociation. The blood sugar concentration of mice injected with the mercaptoethanol-reduced insulin was (5.0 ± 3.2) μmol.L-1, while those injected with the original insulin was (1.4 ± 1.2 ), the difference was significant (Q test, p <0.01). Blood sugar concentration of the mice was 0.3 ± 0.2 μmol.L-1 for the labeled insulin, and was about the same with that for the or The result suggests that while disulfide bonds of polypeptide were reduced by mercaptoethanol, it became free sulfhydryl group, and its bioactivity descended. Then free sulfhydryl group was chelated with 99mTc under mild condition, restablishing the disulfide bond, therefore, the bioactivity came back. The 99mTc-labeled insulin was stable during 24h.