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We developed a technique to assess regional myocardial perfusion by quantifying echocardiographic myocardial contrast appearance and intensity after aortic root injection of an agitated diatrizoate meglumine solution. The technique was validated by comparing digitized echocardiographic contrast parameters to regional perfusion in the circumflex bed determined by calibrated Doppler flow probe and antemortem monastral blue staining. Regional perfusion was altered by circumflex stenosis, occlusion, and reactive hyperemia. Contrast effects were measured in an initial subset of six dogs by peak intensity change, time to peak intensity, maximal rate of intensity rise, and mean intensity change integrated over 1, 2, or 3 seconds after contrast appearance (MI1, MI2, MI3). MI2 and MI3 best predicted regional perfusion (r = 0.93, standard error of the estimate [SEE] 0.38 ml/gm/min for each). These findings were confirmed in a second subset of six dogs (r = 0.84, SEE = 0.70 ml/gm/min). Although there was a relatively broad standard error for the prediction of absolute perfusion for the pooled data, for individual dogs data were internally consistent so that each had r greater than 0.88 for its varied flow states. The hyperemic ratio calculated by contrast echocardiography correlated well with the Doppler value (r = 0.85). Observer and study-to-study predictive variabilities were small (SEE 0.19 to 0.32 ml/gm/min). No alterations were seen in hemodynamics or reactive hyperemia after 25 consecutive injections over a 90-minute period. Contrast echocardiography with aortic root contrast injection tracks changes in regional blood flow. This approach can assess regional coronary reserve and detect changes in regional myocardial perfusion during acute ischemia and drug intervention.