Abstract Objectives Prompt identification of necrosis and apoptosis in the infarct core and penumbra region is critical in acute stroke for delineating the underlying ischemic/reperfusion molecular pathologic events and defining therapeutic alternatives. The objective of this study was to investigate the capability of 99mTc-labeled duramycin in detecting ischemia-reperfusion injury in rat brain after middle cerebral artery (MCA) occlusion. Methods Ischemic cerebral injury was induced in ten rats by vascular insertion of a nylon suture in the left MCA for 3hr followed by 21–24hr reperfusion. After i.v. injection of 99mTc-duramycin (1.0-3.5mCi), dynamic cerebral images were acquired for 1hr in six rats using a small-animal SPECT imager. Four other rats were imaged at 2hr post-injection. Ex vivo images were obtained by autoradiography after sacrifice. Histologic analyses were performed to assess cerebral infarction and apoptosis. Results SPECT images showed that 99mTc-duramycin uptake in the left cerebral hemisphere was significantly higher than that in the right at 1 and 2hr post-injection. The level of radioactive uptake in the ischemic brain varied based on ischemic severity. The average ratio of left cerebral hot-spot uptake to right hemisphere radioactivity, as determined by computerized ROI analysis, was 4.92±0.79. Fractional washout at 1hr was 38.2±4.5% of peak activity for left cerebral hot-spot areas and 80.9±2.0% for remote control areas (P<0.001). Based on triphenyltetrazolium chloride staining and autoradiograph image data, the hotspot uptake may be associated primarily with the ischemic penumbra, in which high apoptotic activity was observed by cleaved caspase-3 immunocytochemical staining. Conclusions 99mTc-duramycin SPECT imaging may be useful for detecting and quantifying ongoing apoptotic neuronal cell loss induced by ischemia-reperfusion injury.