Knowledge of the biomechanical properties of the mitral valve leaflets and their relation to histologic structure is of importance for understanding the leaflet movement characteristics under normal and pathologic conditions, but such knowledge is not yet available. The aim of this study was to determine biomechanical properties of the human anterior mitral leaflet on a microscopic scale. We used scanning acoustic microscopy (SAM) to examine the human anterior mitral leaflet. Sections of fixed human anterior mitral leaflet tissue were obtained from postmortem human anterior mitral leaflets (n = 5). We measured the speed of sound (nu(L)) in each histologic layer in three regions-of-interest (ROIs): these were at the annular edge, at the valve midpoint and close to the free edge. nu(L) varied in the three histologic layers (p < 0.01). It was higher in the fibrous layer (1.76 km/s) compared with the atrial layer (1.75 km/s) and ventricular layer (1.73 km/s). Also, nu(L) differed between positions along the length of the annulus-free edge line (p < 0.01), showing a decline from the annular edge (1.76 km/s) to the free edge (1.73 km/s), both as a whole and also within the atrial and the fibrous layer. These results demonstrate that the fibrous layer is stiffer than the atrial and ventricular layer and that the leaflet as a whole and within the atrial and the fibrous layer is stiffer at the annulus part in comparison with those near the free edge. (E-mail: ).