Calcification of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated bovine pericardium has not been investigated. The objectives of this study were to characterize pericardium before and after glutaraldehyde pretreatment and to study the pathophysiology of mineralization of glutaraldehyde-preserved pericardium. Pericardial protein was approximately 90% collagen, predominantly Type I. Glutaraldehyde incorporation was complete following 24 hours' incubation (151 X 10(-9) mol/mg). Bovine pericardium pretreated in buffered 0.6% glutaraldehyde, implanted subcutaneously in young rats for 24 hours to 112 days, was analyzed chemically (calcium and phosphorus) and morphologically. Mineralization, detected at 48 hours' implantation, was initially associated with pericardial connective tissue cells and later also collagen. Mean calcium content was 114 micrograms/mg at 21 days and 199 micrograms/mg at 112 days. The morphologic features and the kinetics and degree of mineral accumulation in glutaraldehyde-pretreated bovine pericardium were strikingly similar to those previously determined for porcine aortic valve. These results predict that calcification will critically limit the late durability of clinical pericardial bioprostheses and suggest generalized mechanisms of bioprosthetic tissue mineralization which are probably dependent on modification of implant microstructure by glutaraldehyde pretreatment.