BACKGROUND We hypothesized that by using our newly defined method of destroying microbubbles and measuring their rate of tissue replenishment, we could assess the transmural distribution of myocardial perfusion. RESULTS We studied 12 dogs before and after creation of left anterior descending coronary artery stenoses both at rest and during hyperemia (n=62 stages). Microbubbles were administered as a constant infusion, and myocardial contrast echocardiography (MCE) was performed with the use of different pulsing intervals. The video intensity versus pulsing interval plots derived from each myocardial pixel were fitted to an exponential function: y=A(1-ebetat), where A reflects microvascular cross-sectional area (or myocardial blood volume), and beta reflects mean myocardial microbubble velocity. The product A . beta represents myocardial blood flow (MBF). Average values for these parameters were derived from the endocardial and epicardial regions of interest placed over the left anterior descending coronary artery bed. Radiolabeled microsphere-derived MBF was also measured from the same regions. There was poor correlation between radiolabeled microsphere-derived MBF and A-endocardial/epicardial ratios (EER) (r=0.46). The correlation with beta-EER was better (r=0. 69, P<0.01). The best correlation with radiolabeled microsphere-derived MBF-EER was noted with A . beta-EER (r=0.88, P<0. 01). CONCLUSIONS The transmural distribution of myocardial perfusion can be accurately assessed with MCE with the use of our newly described method of tissue replenishment of microbubbles after their ultrasound-induced destruction. In the model studied, an uncoupling of the transmural distribution of MBF and myocardial blood volume was observed during reversal of the MBF-EER.