The authors used glycerol (Gly) as a cross linking agent to treat bovine pericardium (BP) and compared it mechanically (tensile test) and biologically (subcutaneous implantation in rats) with glutaraldehyde (Glu) fixed lonescu-Shiley BP (ISBP). Maximum tensile stress (Stmax) was the same in both groups (n = 12 each), while maximum strain (Snmax) was larger in Gly BP. Calcium content (Ca) was lower and water content of tissue (WC) was higher in implants of Gly BP versus ISBP (n = 9 each). Gly BP appeared more hydrated than ISBP in specimens studied by scanning electron microscopy (SEM). In addition, SEM of three Gly BP mitral valves in sheep showed good growth of endothelial (En) cells. These biomechanical advantages of Gly BP over ISBP suggest that Gly may be a useful substitute for Glu in cross linking bovine pericardial valves.