Background: One of the most important factors responsible for the calcific failure of bioprosthetic heart valves is glutaraldehyde crosslinking. Ethanol (EtOH) incubation after glutaraldehyde crosslinking has previously been reported to confer anticalcification efficacy for bioprostheses. The present studies investigated the anticalcification efficacy in vivo of the novel crosslinking agent, triglycidyl amine (TGA), with or without EtOH incubation, in comparison with glutaraldehyde. Methods: The TGA crosslinking (± EtOH) was used to prepare porcine aortic valves for both rat subdermal implants and sheep mitral valve replacements, for comparisons with glutaraldehyde-fixed controls. Thermal denaturation temperature, an index of crosslinking, cholesterol extraction, and hydrodynamic properties were quantified. Explant endpoints included quantitative and morphologic assessment of calcification. Results: Thermal denaturation temperatures after TGA were intermediate between unfixed and glutaraldehyde-fixed. EtOH incubation resulted in almost complete extraction of cholesterol from TGA or glutaraldehyde-fixed cusps. Rat subdermal explants (90 days) demonstrated that TGA-EtOH resulted in a significantly greater level of inhibition of calcification than other conditions. Thus, TGA-ethanol stent mounted porcine aortic valve bioprostheses were fabricated for comparisons with glutaraldehyde-pretreated controls. In hydrodynamic studies, TGA-EtOH bioprostheses had lower pressure gradients than glutaraldehyde-fixed. The TGA-ethanol bioprostheses used as mitral valve replacements in juvenile sheep (150 days) demonstrated significantly lower calcium levels in both explanted porcine aortic cusp and aortic wall samples compared with glutaraldehyde-fixed controls. However, TGA-EtOH sheep explants also demonstrated isolated calcific nodules and intracuspal hematomas. Conclusions: The TGA-EtOH pretreatment of porcine aortic valves confers significant calcification resistance in both rat subdermal and sheep circulatory implants, but with associated structural instability.