In recent years, several fixation and anti-calcification methods to preserve bioprostheses have been applied clinically. Studies on the effect of different fixation conditions are few in number; thus we performed a systematic ultrastructural investigation on various non-implanted biological valves, and also determined the extent of their calcification in a subcutaneous rat model. Using atomic absorption spectrophotometry, valve calcium contents were measured after subcutaneous implantation for 12 weeks in Wistar rats. All valves tested showed considerable loss of endothelial cover. Independent of fixation conditions, some bioprostheses showed partial destruction of collagen fibers and rearrangement of the extracellular matrix. With regard to calcium content, two valve groups were identified (p <0.05): (i) those with high calcium content, e.g. Toronto-SPV and Intact (40 mg/g dry tissue); and (ii) those with low calcium content, e.g. Mosaic, Freestyle and Hancock-II (<5 mg/g). Low calcium content of the bioprostheses is thought to result from effective anti-calcification treatment. Ultrastructural changes of prosthetic tissue appeared to promote degenerative calcification.