There is considerable interest in calculating regional stresses in the heart. This, in turn, requires complete information on regional material properties which, surprisingly, is not available. The specific aim of this work, therefore, was to determine if transmural differences exist in the mechanical behavior of passive myocardium in the equatorial region of the heart. Thus, we performed in vitro biaxial experiments on 28 thin, rectangular slabs of noncontracting myocardium excised from four regions within canine hearts: the middle portion of the interventricular septum (n = 8), and the inner (n = 5), middle (n = 9) and outer (n = 6) layers of the lateral left ventricular (LV) free wall. There were three major findings. First, an existing three-dimensional constitutive relation described the nonlinear and anisotropic behavior exhibited in the four regions equally well. Second, the anisotropy was similar in each region. Third, there were, however, regional differences in the strain-energy stored by specimens during identical finite deformations. In particular, specimens from inner and outer portions of the LV free wall tended to be stiffer than those from the middle of the LV free wall and septum. These findings, together with previous results on excised epicardium, suggest that the mechanical properties of the heart are qualitatively similar from region to region, but quantitatively different.