Transmural redistribution of myocardial blood flow (MBF) is the earliest sign of myocardial ischemia. We aimed to evaluate the ability of real-time myocardial contrast echocardiography (MCE) combined with dipyridamole stress to quantify the transmural gradient of MBF during graded coronary stenosis. Real-time MCE was performed in 14 open-chest dogs at seven experimental stages: baseline; hyperemia induced by 6-min infusion of dipyridamole; 50%, 75% and 90% reduction of hyperemic flow after constriction in each stage for 10 min; reperfusion for 10 min; and subtotal occlusion of the left anterior descending coronary artery (LAD) for 90 min. We obtained MCE perfusion parameters from subendocardial (A-endo, beta-endo and A x beta-endo) and subepicardial (A-epi, beta-epi and A x beta-epi) layers of the ventricular septum and calculated their transmural gradients (A-EER, beta-EER and A x beta-EER) and systolic wall thickening (SWT). The sensitivity and specificity of each parameter for predicting 75% reduction of hyperemic flow, which was defined as mild myocardial ischemia, were derived by receiver operating characteristic (ROC) curve analysis. No transmural gradients were found at baseline; during maximal hyperemia and 50% reduction of hyperemic flow. beta-endo, A x beta-endo, beta-EER and A x beta-EER decreased significantly when the hyperemic flow was reduced by 75% or more. In contrast, SWT remained unchanged until the hyperemic flow was reduced by 90%. Among all parameters measured, beta-EER and A x beta-EER had the highest and SWT the lowest sensitivity and specificity in predicting mild myocardial ischemia. In conclusion, real-time MCE combined with dipyridamole stress allows for quantification of the transmural gradient of MBF. beta-EER and A x beta-EER are more sensitive than SWT and other MCE parameters in detecting mild myocardial ischemia.