To study the three-dimensional size, shape, and motion of the mitral leaflets and annulus, we surgically attached radiopaque markers to sites on the mitral annulus and leaflets in seven sheep. After 8 days of recovery, the animals were sedated, and three-dimensional marker positions were measured by computer analysis of biplane videofluorograms (60/s). We found that the oval mitral annulus became most elliptical in middiastole. Both leaflets began to descend into the left ventricle (LV) during the rapid fall of LV pressure (LVP), before leaflet edge separation. The anterior leaflet exhibited a compound curvature in systole and maintained this shape during opening. The central cusp of the posterior leaflet was curved slightly concave to the LV during opening. Markers at the border of the “rough zone” were separated by 10 mm during systole. We conclude that coaptation occurs very near the leaflet edges, that the annulus and leaflets move toward their open positions during the rapid fall of LVP, and that leaflet edge separation, the last event in the opening sequence, occurs near the time of minimum LVP.