High-resolution methods for assessing myocardial perfusion in murine models of cardiovascular disease are needed. We hypothesized that regional hypoperfusion could be assessed with ultrahigh-frequency myocardial contrast echocardiography (MCE) and a novel strategy of depot contrast enhancement. MCE was performed with 30-MHz transthoracic imaging 10 seconds and 10 minutes after intravenous administration of microbubbles in control mice, and in mice after left anterior descending coronary artery ligation. MCE was also performed using size-segregated microbubbles. Microbubble behavior in the microcirculation was evaluated with intravital microscopy. In control mice anterior myocardial enhancement was robust at 10 seconds, but left ventricular cavity attenuation precluded evaluation of posterior segments. After 10 minutes, left ventricular cavity signal cleared but myocardial enhancement persisted, permitting analysis of all segments. The degree of enhancement at 10 min was related to microbubble size, implying retention of large microbubbles transiting pulmonary arteriovenous shunts. Intravital microscopy confirmed capillary lodging of large microbubbles. Infarct size by delayed MCE correlated with fluorescent nanospheres (r = 0.94, P < .001). We conclude that complete assessment of regional myocardial perfusion in the mouse heart is possible with high-frequency MCE and a single intravenous contrast agent injection. This technique can be used for characterizing murine models of myocardial infarction and left ventricular remodeling.