BACKGROUND: Direct assessment of the coronary microcirculation has long been hampered by the limited spatial and temporal resolutions of cardiac imaging modalities. OBJECTIVES: The purpose of this study was to demonstrate 3-dimensional (3D) coronary ultrasound localization microscopy (CorULM) of the whole heart beyond the acoustic diffraction limit (<20 µm resolution) at ultrafast frame rate (>1000 images/s). METHODS: CorULM was performed in isolated beating rat hearts (N = 6) with ultrasound contrast agents (Sonovue, Bracco), using an ultrasonic matrix transducer connected to a high channel-count ultrafast electronics. We assessed the 3D coronary microvascular anatomy, flow velocity, and flow rate of beating hearts under normal conditions, during vasodilator adenosine infusion, and during coronary occlusion. The coronary vasculature was compared with micro-computed tomography performed on the fixed heart. In vivo transthoracic CorULM was eventually assessed on anaesthetized rats (N = 3). RESULTS: CorULM enables the 3D visualization of the coronary vasculature in beating hearts at a scale down to microvascular structures (<20 µm resolution). Absolute flow velocity estimates range from 10 mm/s in tiny arterioles up to more than 300 mm/s in large arteries. Fitting to a power law, the flow rate-radius relationship provides an exponent of 2.61 (r2 = 0.96; P < 0.001), which is consistent with theoretical predictions and experimental validations of scaling laws in vascular trees. A 2-fold increase of the microvascular coronary flow rate is found in response to adenosine, which is in good agreement with the overall perfusion flow rate measured in the aorta (control measurement) that increased from 8.80 ± 1.03 mL/min to 16.54 ± 2.35 mL/min (P < 0.001). The feasibility of CorULM was demonstrated in vivo for N = 3 rats. CONCLUSIONS: CorULM provides unprecedented insights into the anatomy and function of coronary arteries at the microvasculature level in beating hearts. This new technology is highly translational and has the potential to become a major tool for the clinical investigation of the coronary microcirculation.