Our previous two articles have shown that glutaraldehyde-fixed bovine pericardium is nearly isotropic, whether fixed without constraints, with tethering, or with pressure. In this study, we have used uniaxial stress during fixation to produce bovine pericardial material with marked tensile anisotropy. Rectangular and cruciate pericardial samples have been mechanically examined after one of four treatments: (i) fixation under 88-kPa uniaxial stress, (ii) fixation under 176-kPa uniaxial stress, (iii) 3 h of 176-kPa uniaxial stress in saline followed by 24-h fixation under the same stress, (iv) fixation under 176-kPa uniaxial stress followed by a second fixation under 176-kPa stress in a direction normal to the first. Strips of material were cut at 0 degree, 30 degrees, 60 degrees, and 90 degrees to direction of the initial stress, and tested for response to cyclic loading, stress relaxation, plastic deformation, and fracture properties. Fixation under uniaxial stress produced anistropy similar to that seen in porcine aortic valve leaflets; however, the overall extensibility of the material depended on the applied stress and the aspect ratio of the stressed sample. While loading in saline produced no change, the sequential biaxial stressing produced a reduction in anisotropy, suggesting exposure of additional crosslinking sites. Uniaxial stress during fixation may be a useful method for construction of anisotropic heart valve leaflets.