Although ultrasound-targeted microbubble destruction (UTMD) has been shown to induce bioeffects, UTMD is still desirable for therapeutic applications. Therefore, we studied the effects of UTMD on perfusion and function of the rat heart, assessed by (99m)Tc-MIBI pinhole-gated SPECT (Ph-gSPECT) compared with biomarker release and histopathology. Fifty-two male Wistar rats were studied. UTMD was performed using SonoVue, with a mechanical index of 1.0 or 1.6. Controls were treated without microbubbles or without ultrasound application. At baseline, day 1, day 7 and day 30, 35 rats were imaged with (99m)Tc-MIBI Ph-gSPECT to quantify left ventricular perfusion and function. In addition, troponin release and histopathology were investigated. No significant differences were observed for left ventricular ejection fractions, end-systolic and end-diastolic volumes, regional perfusion and functional scores up to 30 days after UTMD compared with controls. UTMD induced mild troponin release and early erythrocyte extravasation without necrosis, inflammation or fibrosis. Although UTMD has the potential to induce microlesions of the heart in small animals, these effects were transient without histological evidence of irreversible damage. Furthermore, UTMD does not induce abnormalities on perfusion or function of the heart, as assessed by Ph-gSPECT, which is reassuring concerning the use of SonoVue for potential therapeutic applications. (E-mail: sophie.hernot@gmail.com).