A noncalcifying drug, aminodiphosphonate, was immobilized on pericardial collagen to inhibit calcification. The parameters of ADP binding were optimized by using labeled tracer. The effects of the detergents, Triton X-100 and sodium dodecyl sulfate, pH, and temperature on ADP binding to collagen in fresh pericardium, and stabilization of ADP-collagen bond by borohydride (BH) reduction, were optimized. These studies indicate that pretreatment of pericardium with SDS and TX-100 increases ADP binding three- to fivefold. Three-step cross-linking via Schiff-base reactions between collagen, ADP, and cross-linking via Schiff-base reactions between collage, ADP, and glutaraldehyde gives a high value of 30 to 35 molecules of ADP per collagen in pericardium. Twenty-five millimeter tissue valves were made with detergent (1% SDS, 1% TX-100) treated ADP-bound pericardium and implanted in the mitral annuli in calves, which were killed at 60 days post implantation. Regional calcium deposition was measured with atomic absorption spectrometry. The calcium level (micrograms/mg of tissue) in components of control and four processed valves were determined. Regional platelet deposition on zones of leaflets and components of tissue valves were quantified with 111In-labeled autologous platelets. The calcium levels in thrombus and flexion zones of treated valves are lower than that in the control valve. SEM studies of leaflet surfaces at 60 days indicate that these treatment processes reduce calcification and promote spontaneous cell coverage on leaflets around the smooth surface of the outflow tract.