Myocardial contrast echocardiography (MCE) was used to construct three-dimensional maps of perfusion defects in closed-chest mice, with and without myocardial infarction (MI) induced by permanent coronary ligation. Contiguous, short-axis MCE cine images spanning the heart from apex to base were acquired at 1 mm elevations in each mouse. MCE images at each elevation were color-coded to indicate relative perfusion and were compared with postmortem histology. A strong correlation (R > 0.93) in the size of perfused areas was observed between in vivo measurements and the results of conventional ex vivo tissue staining. 3D multislice and 3D surface renderings of perfusion distribution were created and these perfusion maps also matched well with postmortem histology. These methods provide for the noninvasive determination of the total ischemic region placed at risk by coronary occlusion: this is a critical variable in assessing the potential of novel therapeutic agents to reduce MI size in murine models of ischemia/reperfusion injury.