BACKGROUND: -The ability to noninvasively evaluate murine myocardial blood flow (MBF) in vivo would provide an important tool for cardiovascular research. Myocardial contrast echocardiography (MCE) has been used to measure MBF; however, it has not been validated in mice. This study assesses whether MCE can evaluate MBF at rest and after vasodilation and measure the maximal augmentation (coronary reserve) of MBF in mice. Wild-type (WT) and nitric oxide synthase 3 (NOS3)-deficient (NOS3(-/-)) mice were studied. Methods and Results-MCE was performed at baseline and after intravenous infusion of acetylcholine or adenosine. Definity contrast agent was infused, and parasternal views were acquired in real-time mode. Replenishment curves of myocardial contrast were obtained, and rates of signal rise (beta) and plateau intensity (A) were calculated. MBF estimated by the product of A and beta (Abeta) was compared with that measured with fluorescent microspheres. MCE analysis was feasible in 98% (52/53) of mice. MBF measured by microspheres increased with adenosine and correlated closely with Abeta. There was no difference in MCE-derived MBF between WT and NOS3(-/-) mice at rest. Adenosine infusion increased MBF by 3.0+/-0.6-fold in NOS3(-/-) mice and 2.5+/-0.3-fold in WT (P=0.58 between genotypes). Acetylcholine induced an increase of 2.4+/-0.2-fold in MBF in WT mice but did not increase MBF in NOS3(-/-) mice (P<0.0005 versus WT). Conclusions-MBF, coronary reserve, and vasodilator responses can be evaluated accurately in the intact mouse by MCE. This method demonstrated a preserved coronary response to adenosine but an impaired acetylcholine-induced vasodilation in NOS3(-/-) mice compared with WT mice.