Calcification of bioprosthetic valves, which frequently causes their failure, begins in cell remnants analogous to matrix vesicles of physiologic mineralization. Because the enzyme alkaline phosphatase (AP) is important in normal skeletal mineralization, the authors hypothesized that AP also might be present in bioprosthetic valve tissue and thereby contribute to calcification. AP activity of fresh and glutaraldehyde (GLUT) treated bovine pericardium was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol. After 24 hrs in 0.6% HEPES buffered GLUT and storage for 2 weeks in 0.2% GLUT, considerable AP hydrolytic activity remained relative to that of fresh tissue (Vmax: 24 vs 45 microM reaction product/min/mg tissue protein, respectively), although binding was moderately reduced (KM: 1900 vs 1400 microM substrate, respectively). Light microscopic histochemistry suggested cell oriented AP activity. Ultrastructural examination of GLUT treated tissue demonstrated reaction product along membranes of vascular endothelial cells and fibroblasts, the sites of early calcific deposits in bioprosthetic valves. Thus, AP hydrolytic activity is largely preserved following GLUT treatment of bovine pericardium. These results indicate that the widely held view that GLUT eliminates all metabolic activities of bioprosthetic tissue is inaccurate and suggests that examination of the role of AP and other phosphatases may stimulate approaches for inhibiting calcification.