The imaging appearances of 99Tcm-HL91, a new hypoxic imaging agent, in ischemic myocardium were studied and the value of 99 Tcm-HL91 in the evaluation of regional ischemic viable myocardium was explored. Acute myocardial ischemia models were made by coronary artery legations in 18 rats and randomly divided into 2 groups: 99Tcm-HL91 group and 99Tcm-MIBI group. Evan blue infusion during ischemia and TTC staining after operation were used to delineate the area of ischemic and viable myocardium. The isolated heart was sliced in the short axis and then autoradiography was performed. The electron microscopic examination was also done for the myocardial samples. 99Tcm-HL91 and 99Tcm-MIBI uptake activities (counts/g) were measured in the area of ischemic myocardium (T) and normal myocardium (NT) separately. The uptake ratios of 99Tcm-HL91 and that of 99Tcm-MIBI in ischemic myocardium were calculated as T/NT. It was found that the normal myocardium was blue and ischemic or infarct myocardium was negative with Evans blue in all experiment rats. Both the normal and ischemic myocardium was in red color with TTC staining.In the 99Tcm-HL91 group the ischemic myocardium showed much higher uptake over normal myocardium, that was demonstrated both in the autoradiography and quantitative analysis. The ischemic/normal activity ratios were 1.634±0.354. It was suggested that 99Tcm-HL91 might accumulate in ischemic and viable myocardium, which is helpful in the evaluation of hypoxic but viable myocardium and potentially used as a imaging agent to assess myocardial viability.