The presence and activity of proteolytic enzymes has been investigated in vitro on soluble and insoluble preparations obtained from both unimplanted and implanted glutaraldehyde-treated bovine parietal pericardium. Using detection by colorimetric techniques, soluble preparations were shown to hydrolyze enzyme substrates that are characteristic for trypsin-like proteases, cathepsin-like proteases, and collagenase. As detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gradient gels and gel filtration on Sepharose CL-6B, insoluble (pellet) preparations degraded denatured type I collagen in a time-dependent pattern, producing low-molecular-weight fragments. These activities were partially inhibited by phenylmethylsulfonyl fluoride, N-ethyl maleimide, soybean trypsin inhibitor, para-chloromercuribenzoic acid, or ethylenediaminetetraacetic acid, suggesting the presence of a heterogeneous enzymatic mixture. Insoluble preparations incubated with pure pericardial dermatan sulfate proteoglycan detached the glycosaminoglycan chains from their core protein carrier, producing a digestion pattern similar to Cathepsin C. These findings demonstrate the presence of active proteases in glutaraldehyde-fixed bovine pericardium per se and in explanted pericardial bioprosthetic cardiac valves, an additional factor that might contribute to intrinsic extracellular matrix degeneration in pericardial bioprosthetic devices.