Glutaraldehyde fixation of bioprosthetic tissue has been used successfully for almost 40 years. However, it is generally recognized that glutaraldehyde fixation of bioprostheses is associated with the occurrence of calcification. Accordingly, many efforts have been undertaken to develop techniques for the fixation of bioprostheses, which will not lead to calcification. Here we describe a new improved carbodiimide based cross-linking method. Rather than cross-linking the tissue through its free primary amine groups, these groups were first blocked with butanal and the tissue was then cross-linked by means of carbodiimide activation of tissue carboxylic acid groups followed by a reaction with a poly(propylene glycol)bis 2-(aminopropyl) ether, (Jeffamine trade mark ). It was demonstrated that cross-linked porcine leaflets had a calcification of less than 1mg/g tissue after 8 weeks sub-dermal implantion in rats. Furthermore, aortic wall calcification was reduced to 50mg/g, compared to standard glutaraldehyde fixed tissue, which showed 120mg/g tissue calcification in the 8 weeks calcification model used.