The role of tissue discontinuities in anisotropic impulse propagation was assessed in two-dimensional anisotropic monolayers of neonatal rat myocytes cultured on a growth-directing substrate of collagen. Activation spread and distribution of maximal upstroke rate of rise (V̇max) of the action potential were measured with an optical system using a voltage-sensitive fluorescent dye (RH-327) and a 10×10 photodiode array with a spatial resolution ranging from 7 to 15 μm. Activation maps were compared with the cellular architecture and the distribution of gap junctions obtained from immunostaining the gap junction protein connexin43 (Cx43). Four types of structures were studied: (1) dense cell cultures, (2) cultures with anisotropic intercellular clefts of variable size, (3) discontinuities created by inclusion of nonmyocyte cells, and (4) discontinuities resulting from nonuniform expression of gap junctions. In dense monolayers, activation spread was continuous with microinhomogeneities in both longitudinal and transverse directions. The average cell dimensions in such monolayers were smaller than in adult canine myocardium. However, the degree of cellular anisotropy (length-to-width ratio of 5.3±1.4) and connectivity were similar. The presence of small intercellular clefts (less than one cell in length) did not disturb the general pattern of transverse or longitudinal activation spread, but it was associated with wave front microcollisions during transverse propagation and a concomitant increase of V̇max beyond the cleft. Long intercellular clefts caused discontinuous transverse propagation. Conduction velocity and V̇max decreased significantly at narrow isthmuses formed by closely apposed clefts, rendering such sites susceptible for conduction block. In contrast, V̇max increased when the wave front faced the borders of the clefts. Nonmyocyte cells were electrically connected to myocytes and served as sinks for electrotonic currents, thereby producing localized conduction slowing and a decrease in V̇max. Localized inhomogeneity in Cx43 distribution correlated accurately with circumscribed conduction block and changes in V̇max. Our results provide direct experimental evidence that the cellular structure and gap junction distribution correlate with action potential propagation and distribution of V̇max. We suggest that in tissue with a nonuniform anisotropy, connective tissue separating fiber bundles or sites of inhomogeneous connexin distribution may represent predilective sites for block in transverse direction.