The most common cause of prosthetic heart valve failure is severe regurgitation due to rupture of one or more valve cusps that have become mineralized and rigid. To assess the role of stress and strain in the mineralization initiation, in vitro, reference-grade. essentially lipid-free, glutaraldehyde-tanned bovine pericardium (developed as a standard reference material for bioprosthetic device testing) was exposed to a supersaturated calcium phosphate solution in a high-speed valve tester. The pericardium was tested as sheets constructed into tricuspid valves and dumbbell-shaped tensile-test specimens. and then tested at 720 cycles/min, for up to 18 million cycles. Mineralization was followed using X-ray photography, energy-dispersive X-ray analysis, field-emission scanning electron microscopy, and multiple-attenuated internal reflection infrared spectroscopy. Tensile strength of the tissue was determined using a chemomechanical testing apparatus, Phosphate-based tissue mineralization, first concentrated superficially in an annular distribution at the valve cusp bases, was followed by minimal diffuse calcification within the valve cusps. Small crystalline patches also appeared in the tissue compressed by the valve support rings. Mineralization occurred in a distribution similar to that found in clinically explanted prosthetic heart valves. in areas of maximal tensile and compressive stress and maximal strain. Infrared and Tensile test data combine to show that tissue fatigue due to cyclic loading most likely caused breakage of collagen cross-links. Exposure of superficial broken cross-link sites, and unmasking of hydroxyproline binding sites, of the tissue collagen are thus implicated as the main factors in phosphate-radical nucleation events leading to valve mineralization, overshadowing previously cited contributions from tissue lipids and membranes. Mineralization first occurs at the surface regions subjected to the greatest stress and strain, and begins as amorphous phosphate-rich rather than crystalline calcium-rich deposits.