Background: Although glutaraldehyde fixation is known to reduce immunogenicity and degeneration of heart valve bioprostheses, some degree of immunogenicity persists, which may trigger calcification. The aims of this study were to: (1) define the role of alpha-1,3-galactosyltransferase (α-Gal) antigen in valve calcification by comparing α-Gal-positive and α-Gal-deficient (GT-KO) pig pericardium; and (2) elucidate the role of human anti-Gal antibodies in the process of calcification and to determine the potential influence of different tissue-fixation techniques. Methods: Glutaraldehyde-treated pericardium from α-Gal-positive and GT-KO pigs, with or without pre-labeling with human anti-Gal antibodies, were implanted in rats during 1 month. Results: In glutaraldehyde-fixed pericardium, calcification levels were significantly lower in GT-KO pig pericardium (132.8 ± 5.8 μg/mg) as compared with α-Gal-positive pig pericardium (155.7 ± 7.1 μg/mg) (p < 0.015). In glutaraldehyde-fixed pig pericardium followed by a mix of formaldehyde, ethanol and Tween 80 (FET), the calcification levels were lower in GT-KO pig pericardium (0.35 ± 0.1 μg/mg) as compared with α-Gal-positive pig pericardium (4.6 ± 4.2 μg/mg). In glutaraldehyde-fixed pig pericardium + FET pre-incubated with human anti-Gal antibodies, calcification levels were significantly greater in α-Gal-positive pig pericardium (43.8 ± 8.5 μg/mg) as compared with GT-KO pig pericardium (5.7 ± 2.9 μg/mg) (p < 0.0001). Conclusions: This study demonstrates the role of α-Gal antigen and human α-Gal antibodies in the calcification process of valvular bioprostheses. It is suggested that GT-KO pig pericardium could be beneficial as a new source of material for heart valve bioprostheses. © 2010 International Society for Heart and Lung Transplantation.