Previously, we showed that intercellular uncoupling through gap junctions is an important mechanism for maintaining transmural heterogeneities of repolarization that are responsible for ventricular arrhythmias in a broad range of disease models including long QT syndrome and heart failure. However, the complex fiber geometry associated with rotational anisotropy between transmural muscle layers may also influence coupling. To determine the effect of roational anisotropy on transmural coupling, a numerical 3-D model of passive cardiac tissue was developed in which rotational anisotropy was varied in a controlled fashion. Simulations of optical mapping demonstrated that spatial averaging produced a voltage decay in space best fit by a single decaying exponential compared to the theoretically predicted decay. As fiber orientation varied by 90° with respect to the transmural surface, the effective transmural space constant (_TM) changed by only 0.31% in simulations. In contrast, reducing intercellular conductivity by 24% decreased _TM by 7.7%. In the canine wedge preparation (n=5) measured by optical mapping of the epicardial and subepicardial surface was similar transverse (_TV = 0.73 ± 0.10 mm) and transmural (_TM = 0.70 ± 0.08mm) to subepicardial fibers. We confirmed previous findings that _TM in subepicardial layers was significantly reduced by 14 ± 2% compared to deeper layers of myocardium providing evidence for transmural uncoupling in the epi-midmyocardial interface. Conclusions: These data establish the theoretical and experimental basis for measuring intercellular coupling between muscle layers spanning the ventricular wall with optical mapping techniques. Furthermore, this study demonstrates transmural uncoupling at the epicardial-midmyocardial interface may be attributable to heterogeneous expression of cardiac gap junctions not rotational anisotropy.