Carbodiimide cross-linking of bioprosthetic materials has been shown to provide tissue stabilization equivalent to that of glutaraldehyde cross-linking, but without the risk of the release of unreacted or depolymerized cytotoxic reagent after implantation. In this study, the effects of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) treatment on acellularized ovine carotid arteries were studied under two different pH conditions: (i) pH controlled at an optimal value of 5.5; and (ii) a simpler, but industrially significant, uncontrolled pH system. A multimode approach was employed involving biochemical assays, thermomechanical, tensile, and shear mechanical testing, and in vitro enzyme degradation analyses. EDC treatment decreased the hoop tangent modulus of acellular matrix (ACM) arterial grafts measured at 20 kPa of stress regardless of pH control. Extensibility of ACM arterial grafts measured at 20 kPa of stress was reduced after EDC treatment with pH control only. In contrast, shear stiffness of ACM arterial grafts increased to a greater degree under cross-linking without pH control (21 x compared to 14 x with pH control). Thermomechanical analyses revealed that EDC cross-linking with pH control also increased the collagen denaturation temperature of ACM arteries to a greater degree (a rise of 24.3 +/- 0.6 degrees C vs. 21.7 +/- 0.7 degrees C for no pH control), whereas cross-linking without pH control consumed a larger amount of lysine residues after 3 h of treatment. Most interestingly, both EDC treatments were equally effective in stabilizing ACM arteries against multiple degradative enzymes in vitro. The observed differences between EDC treatments under different pH conditions are attributed to differences in the location and types of the exogenous cross-links formed. The absence of pH control may have favored the formation of interfibrillar or intermolecular cross-links in collagen as well as involvement of other extracellular matrix components (proteoglycans and glycosaminoglycans). Furthermore, it may be emphasized that the location or type of cross-links differentially affected the mechanical behavior of treated materials without affecting the increase in resistance to enzymatic degradation. Copyright 2001 John Wiley & Sons, Inc.