Calcification frequently causes failure of bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-pretreated porcine aortic valve or bovine pericardium. Systemic diphosphonate therapy inhibits this disease process, but with adverse effects on overall growth, bone development, and calcium metabolism. The present study was designed to examine the hypothesis that the immobilization of ethanehydroxydi-phosphonate (EHDP) within BHV as the poorly soluble Ca2+ salt would inhibit calcification at drug levels insufficient to produce side effects. Glutaraldehyde-pretreated pericardial BHV tissue was exposed to a physiologic concentration of Na2EHDP (0.14M in 0.05M HEPES, pH 7.4) and subsequently washed in a NaCl or CaCl2 (0.14M in 0.05M HEPES, pH 7.4) solution to precipitate the Na or Ca2+ salts of EHDP on/ within the tissue, respectively. Incorporation of CaEHDP into BHV ranged from 74.8 nM/mg (after 1 h, 37 °C) to 353 nM/mg (2 weeks, 22 °C). None of the Na2EHDP was incorporated into BHV without exposure to CaCl2. In vitro release of CaEHDP from BHV into a physiologic buffer not containing Ca2+ was rapid, with >95% removed after 4 d, while release of CaEHDP into buffer containing a physiologic concentration of Ca2+ ion (1.5 mM) was markedly reduced, with 30% of the precipitated CaEHDP remaining immobilized on or within the tissue matrix following 21 d. Subdermal implants of CaEHDP-pretreated BHV tissue in rats (21-d old, male, CD strain) had significantly less calcification than untreated tissue following 21 d (Ca2+ 2.59 ± 0.264 μg/mg, control 103 ± 10.8 μg/mg) and 60 d (Ca2+ 41.0 ± 11.1 μg/mg, control 150 ± 23.0 μg/mg), and explanted pretreated tissue from the 21- and 60-d studies showed no calcification by microscopic examination. The BHV that were pretreated with the Na2EHDP solution and subsequently washed in either a NaCl or CaCl2 solution did not undergo any detectable morphologic changes. No abnormalities in serum calcium levels, femur development, or overall weight gain were noted. It is concluded that CaEHDP preincorporation into BHV effectively inhibited calcification without adverse systemic effects. However, rapid time-dependent efflux of CaEHDP from the pericardial tissue may limit its usefulness in long-term heart valve replacements.