Calcification of valvular bioprostheses in children is a major problem that has stimulated extensive research in our laboratory. In previous reports, we have shown that the rate of calcification could be reduced by decreasing the phosphate content in the tissue or by blocking calcification binding sites with Mg++ and/or with a surfactant (Tween 80). Since then, we have systematically investigated incorporation of numerous other surfactants and of polyacrylamide within the tissue, and these investigations form the basis of this report. The methods of investigation included subcutaneous implantation of treated tissues in growing rabbits, stability tests, and intracardiac implantation in sheep. Results showed that surfactants differed in their efficacy in mitigating calcification, with N-lauroylsarcosine and triton X-100 being the most efficient. Polyacrylamide incorporation was also efficient in calcification mitigation, but this effect was lost after flexibility testing in vitro or implantation in vivo, a drawback that suggests further research is necessary into stabilization of this polymer. This report not only brings attention to new alternatives in calcification mitigation treatment of bioprosthetic tissues but also underlines important points of methodology.