Objective To evaluate the anatomic localization and size of acute necrotic myocardium in the ischemic-reperfused rat hearts using ^99mTC-Glucarate and microSPECT/CT. Methods The ischemic-reperfused (IR) rat heart models were established by ligating left anterior descending coronary artery for 60 min. ^99mTC-Glucarate was intravenously injected into the rats 24 hours after IR operations. Images were acquired 30 min after administration of ^99mTC-Glucarate using microSPECT/CT. Anatomic localization and size of acute necrotic myocardium were analyzed with microSPECT/CT imaging,and these results were compared to those determined by triphenyltetrazolium chloride (TTC) staining. Results The microSPECT/CT images showed hot spot accumulations of ^99mTC-Glucarate in IR hearts (the heart-to-liver ratio was 1.90±0.33), not in controls (P < 0.05). The anatomic localization of ^99mTC-Glucarate-labeled necrotic myocardium were in correspondence with TTC staining results. The hot spot size was related significantly to necrotic myocardial size determined by TTC staining (R²=0.964). Conclusions The localization and size of acute necrotic myocardium can be assessed by non-invasive microSPECT/CT imaging with ^99mTc-Glucarate.