Quantification of regional myocardial tissue blood flow (RMBF) based on contrast echocardiography has yet to be achieved. This study validated our recently proposed algorithm for quantification of RMBF with colored microspheres. Experiments were carried out in an isolated rabbit heart preparation (n = 11). Aortic root injections of perfluoropropane-filled albumin microsphere solution (FS069) and colored microspheres were performed at five levels of coronary flow achieved by altering perfusion pressure. During each injection of contrast material, consecutive end-diastolic images of the heart and an extracardiac reference chamber were acquired with a 7.5 MHz transducer and digitized. Time-intensity curves from the reference chamber and myocardial regions of interest, corresponding to the anatomic segments used for colored microsphere analysis, were analyzed for RMBF. Blood flow was calculated as the intravascular volume fraction (ratio of areas under myocardial and reference curves) divided by mean transit time (deconvolution of impulse response) and compared with those obtained with colored microspheres. Injections of FS069 resulted in highly reproducible enhancement of myocardial contrast. Analysis of time-intensity curves provided consistent measurements of RMBF (r = 0.91), which correlated highly with microsphere data (r = 0.84). The use of this new algorithm allows accurate quantification of RMBF in the isolated heart model. Further validation of this approach in an animal model with peripheral intravenous injections of contrast material will allow noninvasive clinical measurements of RMBF.