Although previous studies report a reduction in myocardial volume during systole, myocardial volume changes during the cardiac cycle have not been quantitatively analyzed with high spatiotemporal resolution. We studied the time course of myocardial volume in the anterior mid-left ventricular (LV) wall of normal canine heart in vivo (n=14) during atrial or LV pacing using transmurally implanted markers and biplane cineradiography (8msec/frame). During atrial pacing, there was a significant transmural gradient in maximum volume decrease (4.1%, 6.8% and 10.3% at subepi, midwall and subendo layer, respectively, p=0.002). The rate of myocardial volume increase during diastole was 4.7±5.8, 6.8±6.1 and 10.8±7.7 mL/min/g, respectively, which is substantially larger than the average myocardial blood flow in the literature measured by the microsphere method (0.7-1.3 mL/min/g), assuming that the blood inflow occurs only during diastole. In the early activated region during LV pacing, myocardial volume began to decrease prior to the LV pressure upstroke. We conclude that the volume change is greater than would be estimated from the known average transmural blood flow. This implies the existence of blood-filled spaces within the myocardium, which could communicate with the ventricular lumen. Our data in the early activated region also suggest that myocardial volume change is caused not by the intramyocardial tissue pressure but by direct impingement of the contracting myocytes on the microvasculature.