Measurements of coronary microvascular parameters in situ are difficult because of the thickness of the heart muscle and cardiac contraction. Both of these problems hamper the visualization of the coronary microcirculation. We have refined methodological approaches that enable the study of the coronary microcirculation in situ. In the first approach, microvessels can be visualized in the beating heart using a preparation that compensates for cardiac motion by creating an illusion that the heart is motionless. This is accomplished by flashing a stroboscopic light source once per heart cycle at the same point in each cycle and synchronizing a ventilator with the cardiac cycle. Images of microvessels can be obtained using standard intravital video-microscopic techniques. To visualize the intramural and subendocardial microcirculation, studies are completed in isolated hearts. In this preparation, measurements of microvascular diameters and pressures can be performed in both the subepicardial and subendocardial microcirculations. This latter approach allows insight into transmural differences of coronary microvascular regulation.