Objective:To study 99Tcm-MPO ([99mTc N (MPO) (PNP5)+: 2-mercapto pyridine N-oxide and N-ethoxy ethyl-N, N-two [2 (two (3-methoxy propyl) phosphine) ethyl] amine) biodistribution in normal mice,and to study the biodistribution in vivo and its application as a new myocardial perfusion imaging tracer on the clinical application value. Methods:99Tcm-MPO was synthesized in two steps, and HPLC method was performed to analyze the radiolabeling rate and in vitro stability of 99Tcm-MPO. The normal Kunming rats as experimental group received injection of 99Tcm-MPO 3.7 MBq via the tail vein, while the control group was injected with 99Tcm-MIBI 3.7 MBq, and the biodistribution (%ID/g) in dissected organs were measured respectively at 5, 15, 30, 60 and 120 min post-injection (p.i.). Two normal New Zealand rabbits were intravenously injected with 99Tcm-MPO and 99Tcm-MIBI 37MBq at the ear margin, and static images were performed by SPECT at 5, 15, 30, 60 min p.i., respectively. We respectively proceed SPECT/CT imaging at 15 and 60 min p.i. after blocking blood flow with a balloon in the experimental miniature pigs＇ coronary artery left circumflex branch. Results:99Tcm-MPO had high radiolabeling yield of (98.8 ± 1.0)% and radiochemical purity after 12 h of (96.5 ± 0.8)% in vivo. In vivo biodistribution study demonstrated that uptake of 99Tcm-MPO in the kidneys was similar to 99Tcm-tetrofosmin at 5 min p.i.. The uptake of 99Tcm-MPO in liver was(30.38 ± 0.43)% ID/g at 5 min, slightly more than 99Tcm-MIBI and 99Tcm-tetrofosmin, but 99Tcm-MPO has a faster liver clearance. The uptake of 99Tcm-MPO in the heart was (9.38 ± 0.70)%ID/g at 15min p.i., slightly lower than 99Tcm-MIBI, but there was no significant difference. The factor that 99Tcm-MPO has a significantly higher heart/liver ratios than 99Tcm-MIBI and 99Tcm-tetrofosmin at 15min p.i. (heart/liver ratios: 2.21 ±0.44 vs. 0.62 ± 0.02 for 99Tcm-MIBI and 0.89 ± 0.06 for 99Tcm-tetrofosmin, F=22.29, P=0.016). The rabbits planar imaging after intravenous injection 99Tcm-MPO showed visible images of the heart and liver at 5 min p.i., and the radioactivity uptake in liver significantly reduced at 15 min p.i.. 99Tcm-MPO has higher heart-liver ratios than 99Tcm-MIBI and 99Tcm-tetrofosmin at 15min p.i. (heart-liver ratios: 0.85 ±0.32 vs. 0.71 ± 0.15 for 99Tcm-MIBI and 0.59 ± 0.64 for 99Tcm-tetrofosmin), but there was no significant difference. SPECT/CT showed decreased uptake of 99Tcm-MPO by lesions with ischemia and reperfusion injury in porcine models, and there was no significant difference. Conclusion:99Tcm-MPO is a new myocardial perfusion imaging tracer. Compared with 99Tcm-MIBI,99Tcm-MPO showed higher uptake in heart, longer myocardial radioactivity retention time, and quicker liver clearance. The heart-liver radioactivity uptake was higher than 99Tcm-MIBI and 99Tcm-tetrofosmin at 15min p.i.. 99Tcm-MPO may be used for early myocardial perfusion imaging, and has a good prospect of clinical application.