We measured leaflet displacements and used inverse finite element analysis to define, for the first time, the material properties of mitral valve (MV) leaflets in vivo. Sixteen miniature radiopaque markers were sewn to the MV annulus, 16 to the anterior MV leaflet, and one on each papillary muscle tip in 17 sheep. 4-D coordinates were obtained from biplane videofluoroscopic marker images (60f/s) during three complete cardiac cycles. A finite element model of the anterior MV leaflet was developed using marker coordinates at the end of isovolumic relaxation (IVR, when pressure difference across the valve is approximately zero), as the minimum-stress reference state. Leaflet displacements were simulated during IVR using measured left ventricular and atrial pressures. The leaflet shear modulus (G_circ-rad) and elastic moduli in both the commisure-commisure (E_circ) and radial (E_rad) directions were obtained using the Method of Feasible Directions to minimize the difference between simulated and measured displacements. Group mean (±SD) values (17 animals, 3 heartbeats each, i.e., 51 cardiac cycles) were: G_circ-rad= 121±22 N/mm²; E_circ= 43±18 N/mm²; and E_rad= 11±3 N/mm² (E_circ > E_rad, p<0.01). These values, much greater than those previously reported from in vitro studies, may result from activated neurally-controlled contractile tissue within the leaflet that is inactive in excised tissues. This could have important implications, not only to our understanding of mitral valve physiology in the beating heart, but for providing additional information to aid the development of more durable tissue-engineered bioprosthetic valves.