The effectiveness of ethanol pretreatment on preventing calcification of glutaraldehyde-fixed porcine aortic bioprosthetic heart valve (BPHV) cusps was previously demonstrated, and the mechanism of action of ethanol was attributed in part to both lipid removal and a specific collagen conformational change. In the present work, the effect of ethanol pretreatment on BPHV aortic wall calcification was investigated using both rat subdermal and sheep circulatory implants. Ethanol pretreatment significantly inhibited calcification of BPHV aortic wall, but with less than complete inhibition. The maximum inhibition of calcification of BPHV aortic wall was achieved using an 80% ethanol pretreatment; calcium levels were 71.80 ± 8.45 g/mg with 80% ethanol pretreatment compared to the control calcium level of 129.90 ± 7.24 g/mg (p = 0.001). Increasing the duration of ethanol exposure did not significantly improve the inhibitory effect of ethanol on aortic wall calcification. In the sheep circulatory implants, ethanol pretreatment partly prevented BPHV aortic wall calcification with a calcium level of 28.02 ± 4.42 g/mg compared to the control calcium level of 56.35 ± 6.14 g/mg (p = 0.004). Infrared spectroscopy (ATR-FTIR) studies of ethanol-pretreated BPHV aortic wall (vs. control) demonstrated a significant change in protein structure due to ethanol pretreatment. The water content of the aortic wall tissue and the spin-lattice relaxation times (T 1 ) as assessed by proton nuclear magnetic resonance spectroscopy did not change significantly owing to ethanol pretreatment. The optimum condition of 80% ethanol pretreatment almost completely extracted both phospholipids and cholesterol from the aortic wall; despite this, significant calcification occurred. In conclusion, these results clearly demonstrate that ethanol pretreatment is significantly but only partially effective for inhibition of calcification of BPHV aortic wall and this effect may be due in part to lipid extraction and protein structure changes caused by ethanol. It is hypothesized that ethanol pretreatment may be of benefit for preventing bioprosthetic aortic wall calcification only in synergistic combination with another agent.