Glutaraldehyde-crosslinked bioprosthetic heart valves (BPHV) often fail due to calcification-related deterioration. Ethanol pretreatment of BPHV prevents calcification in animal models. The present studies were undertaken to further understand the mechanisms of ethanolic prevention of calcification. We show that other alcohols such as methanol and isopropanol also prevent BPHV calcification in a rat subdermal model. Furthermore, alcohol pretreatments during glutaraldehyde crosslinking were ineffective in preventing BPHV calcification. Storage of ethanol-pretreated BPHV in glutaraldehyde allowed partial return of the calcification. Therefore, storage in alcoholic glutaraldehyde is the best method of preserving the anti-calcification effects of ethanol.