Stress echocardiography is an important screening test for coronary artery disease. Currently, cardiologists rely on visual analysis of left ventricular (LV) wall motion abnormalities, which is subjective and qualitative. We previously used finite-element models of the regionally ischemic left ventricle to develop a wall motion measure, 3DFS, for predicting ischemic region size and location from real-time 3D echocardiography (RT3DE). The purpose of this study was to validate these methods against regional blood flow measurements during regional ischemia and to compare the accuracy of our methods to the current state of the art, visual scoring by trained cardiologists. We acquired RT3DE images during 20 brief (<2 min) coronary occlusions in dogs and determined ischemic region size and location by microsphere-based measurement of regional perfusion. We identified regions of abnormal wall motion using 3DFS and by blinded visual scoring. 3DFS predicted ischemic region size well (correlation r 2 = 0.64 against microspheres, p < 0.0001), reducing error by more than half compared to visual scoring (8 ± 9% vs. 19 ± 14%, p < 0.05), while localizing the ischemic region with equal accuracy. We conclude that 3DFS is an objective, quantitative measure of wall motion that localizes acutely ischemic regions as accurately as wall motion scoring while providing superior quantification of ischemic region size. © 2010 Biomedical Engineering Society.