Objective To evaluate the feasibility and accuracy of measurement of myocardial perfusion defects with intravenous contrast-enhanced real-time three-dimensional echocardiography （CE-RT3DE）. Methods RT3DE was performed in 21 open-chest mongrel dogs undergoing acute ligation of the left anterior descending artery （LAD

n=14） or distal branch of the left circumflex artery （LCX

n=7）. A perfluorocarbon microbubble contrast agent was injected intravenously to assess the resulting myocardial perfusion defects with Philips Sonos-7500 ultrasound system. Evans blue dye was injected into the occluded coronary artery for subsequent anatomic identification of underperfused myocardium. In vitro anatomic measurement of myocardial mass after removal of the animal’s heart was regarded as the control. Blinded off-line calculation of left ventricular mass and perfusion defect mass from RT3DE images were performed using an interactive aided-manual tracing technique.Results Total left ventricular （LV） myocardial mass ranged from 38.9 to 78.5 （mean±SD: 60.0±10.1） g. The mass of perfusion defect ranged from 0 to 21.4 （mean±SD: 12.0±5.0） g or 0 to 27% of total LV mass （mean±SD: 19%±6%）. The RT3DE estimation of total LV mass （mean±SD: 59.8±9.9 g） strongly correlated with the anatomic measurement （r=0.98; y=2.01+0.96x）. The CE-RT3DE calculation of the mass of underperfused myocardium （mean±SD: 12.3±5.3 g） also strongly correlated with the anatomic measurement （r=0.96; y=-0.10+1.04x） and when expressed as percentage of total LV mass （r=0.95; y=-0.20+1.04x）. Conclusions RT3DE with myocardial contrast opacification could accurately estimate underperfused myocardial mass in dogs of acute coronary occlusion and would play an important role in quantitative assessment of myocardial perfusion defects in patients with coronary artery disease.