Optical mapping has been used to determine changes in transmembrane voltage during electrical stimulation pulses (deltaVm) and whether deltaVm depends on fiber orientation, as predicted from bidomain models. Fiber orientation in an approximately 1 cm2 mm mapped region on the rabbit left or right ventricular epicardium was estimated optically from the fast axis of action potential (AP) propagation. Hearts were paced outside of the region to produce APs. Unipolar stimulation (S2) was then applied early in the AP, when tissue was refractory, so that deltaVm was not obscured by a new AP. Anodal S2 produced negative deltaVm near a point S2 electrode and away from it in the direction perpendicular to the fibers. Anodal S2 produced reversal of the sign of deltaVm about 1 mm from the electrode in the direction parallel to the fibers, such that a positive deltaVm existed about 1-5 mm away from the electrode. Reversal of the sign of deltaVm in the direction parallel to the fibers also occurred with cathodal S2, which produced a negative deltaVm away from the electrode parallel to the fibers. The results indicate a "dogbone" pattern of deltaVm, as predicted from bidomain models that have resistance anisotropy ratios of trabecular muscles (ie, an intracellular ratio that does not equal the extracellular ratio). Thus, optical mapping can indicate fiber orientation and deltaVm, and the deltaVm during unipolar stimulation reverses sign on the axis parallel to the fibers, which differs from one-dimensional model predictions. The deltaVm agrees with multidimensional bidomain model predictions that have unequal resistance anisotropy.