A new acellular, natural, biodegradable matrix has been discovered in the cholecyst-derived extracellular matrix (CEM). This matrix is rich in collagen and contains several other macromolecules useful in tissue remodeling. In this study, the principal material axes, collagen fiber orientations, and biaxial mechanical properties in a physiological loading regime were characterized. Fiber direction was determined by polarized light microscopy, and the principal axes and degree of anisotropy were determined mechanically. Macroscopic equibiaxial strain tests were then conducted on preconditioned specimens. While 13% of the area of CEM contains collagen fibers oriented between 50 degrees and 60 degrees from the neck-fundus axis, the principal material axis was oriented 63 degrees +/- 13.7 degrees, with an aspect ratio of 0.11 +/- 0.06, indicating a weak anisotropy in that direction. Under biaxial loading, CEM exhibited a large toe region followed by an exponential rise in stress in both principal and perpendicular axis directions, similar to other materials currently under research. There was no significant difference between the biaxial stress-strain profile and the burst stress-strain profile. The results demonstrate that CEM is weakly anisotropic and it has the ability to support large strains across a physiological loading regime. (c) 2007 Wiley Periodicals, Inc.