To date, there are no constitutive models for either the natural or bioprosthetic aortic valve (AV), in part due to experimental complications related to the AV's small size and heterogeneous fibrous structure. In this study, we developed specialized biaxial testing techniques for the AV cusp, including a method to determine the local structure-strain relationship to assess the effects of boundary tethering forces. Natural and glutaraldehyde (GL) treated cusps were subjected to an extensive biaxial testing protocol in which the ratios of the axial tensions were held at constant values. Results indicated that the local fiber architecture clearly dominated cuspal deformation, and that the tethering effects at the specimen boundaries were negligible. Due to unique aspects of cuspal fiber architecture, the most uniform region of deformation was found at the lower portion as opposed to the center of the cuspal specimen. In general, the circumferential strains were much smaller than the radial strains, indicating a profound degree of mechanical anisotropy, and that natural cusps were significantly more extensible than the GL treated cusps. Strong mechanical coupling between biaxial stretch axes produced negative circumferential strains under equibiaxial tension. Further, the large radial strains observed could not be explained by uncrimping of the collagen fibers, but may be due to large rotations of the highly aligned, circumferential-oriented collagen fibers in the fibrosa. In conclusion, this study provides new insights into the AV cusp's structure-function relationship in addition to requisite data for constitutive modeling.