Intravascular transplantation of bone marrow-derived mesenchymal stem cells (MSCs) is a promising therapeutic approach after acute myocardial infarction. Efficacy and targeting of myocardial cell engraftment are crucial variables determining the therapeutic value of MSC transplantation. Highly focused ultrasound-mediated stimulation of microbubbles (hf-UMS) allows locoregional pre-treatment of target tissue. In a "proof of concept" study, we investigated augmentation of site-targeted MSC engraftment with hf-UMS. We further evaluated the ability of transplanted MSCs to transmigrate across the endothelial barrier into non-ischemic and post-ischemic myocardium in vivo. After acute myocardial ischemia and reperfusion, rats received hf-UMS focused on the anterior left-ventricular wall followed by intravascular transplantation of MSCs. Global and regional myocardial engraftment of MSCs was quantified by means of confocal laser-scanning microscopy; endothelial adhesion, transendothelial migration and invasion of basement membrane were distinguished. Targeted myocardium exhibited higher amount of transplanted MSCs vs. non-targeted tissue. The rate of transendothelial migration was lowest in non-ischemic (41.2+/-2%) compared to post-ischemic myocardium (53+/-5.7%, p<0.01). Hf-UMS significantly increased the transmigration rate to 50+/-6.1% (p<0.05) and 64+/-8.9% (p<0.05), respectively. Additionally, myocardial segments exposed to hf-UMS revealed an onset of protease activity. Signs of undesired biological effects, such as induction of apoptosis and/or myocardial necrosis were not observed. This study provides the first evidence of the migration of MSCs across the myocardial endothelium in vivo. Hf-UMS not only improves myocardial engraftment of MSCs but also allows locoregional targeting of post-ischemic myocardium.