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Mitral and aortic valves are known to be coupled via fibrous tissue connecting the two annuli. Previous studies evaluating this coupling have been limited to experimental animals using invasive techniques. The new matrix array transesophageal transducer provides high-resolution real-time 3D images of both valves simultaneously. We sought to develop and test a technique for quantitative assessment of mitral and aortic valve dynamics and coupling.Matrix array transesophageal (Philips iE33) imaging was performed in 24 patients with normal valves who underwent clinically indicated transesophageal echocardiography. Custom software was used to detect and track the mitral and aortic annuli in 3D space throughout the cardiac cycle, allowing automated measurement of changes in mitral and aortic valve morphology. Mitral annulus surface area and aortic annulus projected area changed reciprocally over time. Mitral annulus surface area was 8.0±2.1 cm2 at end-diastole and decreased to 7.7±2.1 cm2 in systole, reaching its maximum (10.0±2.2 cm2) at mitral valve opening. Aortic annulus projected area was 4.1±1.2 cm2 at end-diastole, then increased during isovolumic contraction reaching its maximum (4.8±1.3 cm2) in the first third of systole and its minimum (3.6±1.0 cm2) during isovolumic relaxation. The angle between the mitral and aortic annuli was maximum (136±13°) at end-diastole and decreased to its minimum value (129±11°) during systole.This is the first study to report quantitative 3D assessment of the mitral and aortic valve dynamics from matrix array transesophageal images and describe the mitral-aortic coupling in a beating human heart. This ability may have impact on patient evaluation for valvular surgical interventions and prosthesis design.