The aim of this study was to detect biologic factors in the structural deterioration of bioprosthetic heart valves. Prostheses were removed from patients after 4-8 years of implantation and submitted to biochemical and morphologic assays. Successive staining of biologic sections revealed colocalization of lipids and glycosaminoglycans underneath calcifications in the disintegrated extracellular matrix. On biochemical assays, the amidolysis of synthetic peptide substrates indicated thrombin, plasmin, and tissue plasminogen activator activities in the nonhemocompatible leaflets; 0.15 mol NaCl, 0.05 mol Tris, and 5 mmol CaCl2 extracts from the prostheses cleaved the peptide substrate for collagenase and lysed gelatin gels. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate disclosed the presence of low molecular mass polypeptides in extracts of the deteriorated prostheses. The detection of plasmin and collagenolytic enzyme(s), and the known broad proteolytic activity of plasmin, may point to the role of activation of the fibrinolytic system in the proteolytic degradation of bioprosthetic valves.