Calcification of bioprostheses used for heart valve replacement is a serious problem, since it causes bioprosthetic dysfunction. In vivo, bioprostheses are subjected to large mechanical stresses during each cardiac cycle. We investigated whether stresses play a major role in calcification of bioprostheses. Previous studies of Carpentier-Edwards porcine, Hancock porcine, and Ionescu-Shiley pericardial bioprostheses indicated that the highest stresses occurred in the areas of greatest flexion of the leaflet. In porcine bioprostheses, stresses were greater in the commissural region than at the base, and were compressive on the aortic surface of the leaflet. The pericardial tissue showed shear deformation in the zone of flexion. In the present study, the three types of bioprostheses were implanted in the aortic position in calves to investigate the development, location, and distribution of calcification. Visual, radiographic, and histologic techniques were used. All bioprostheses showed calcification which began in the area of leaflet flexion. In porcine bioprostheses, calcification occurred earlier in the commissural region than at the base. The earliest calcific deposits were localized within collagen cords on the aortic surface of the leaflets. In pericardial bioprostheses, calcification occurred at multiple foci along the zone of leaflet flexion and was located between and within layers of collagen along planes parallel to the leaflet surface. Hence calcification in all bioprostheses began in the areas of greatest stress. In porcine bioprostheses, calcification was present where collagen fibers are likely to have been damaged by compressive stresses. In pericardial bioprostheses, calcification was found along the planes of shear where structural integrity is likely to have been disrupted by the sliding of individual layers of collagen over each other. It is concluded that mechanical stresses initiate calcification by damaging the structural integrity of the leaflet tissue. Therefore, calcification of bioprostheses can be inhibited by reducing functional stresses through the modification of design and tissue properties to duplicate those of the natural aortic valve.