Microscopic discontinuities in electrical activation were assessed in synthetic strands of neonatal rat myocytes cultured on a growth-directing matrix. An optical method using voltage-sensitive dye (RH-237) and a photodiode technique was used for recordings of membrane potential changes with subcellular resolution. Spatial resolution of the method (diameter of measurement area, 5.5 μm; interdiode distance, 30 μm) allowed for simultaneous measurements of cytoplasmic conduction time within a single cell and junctional conduction time across the cell border. In one-dimensional cell chains, where cells were juxtaposed by end-to-end connections but devoid of lateral connections, propagation of the excitation wave was strongly nonuniform: cytoplasmic conduction time was 38±30 (mean±SD) microseconds (n=37), whereas junctional conduction time was 118±40 microseconds (n=27, P<.0001). A mean delay introduced by a single junction was 80 microseconds, or 51% of conduction time. In two-dimensional strands consisting of several cells in width, which exhibited lateral as well as end-to-end connections, inhomogeneity of conduction was smaller: the cytoplasmic and junctional conduction times were 57±30 (n=46) and 89±40 (n=48) microseconds, respectively (P<.0001); mean junctional conduction delay was 32 microseconds (22% of conduction time). Mathematical modeling suggested that the averaging effect of lateral connections is caused by lateral convergence of local excitatory current beyond and lateral divergence before end-to-end connections. Our results demonstrate that the current flow through lateral cell-to-cell connections smooths the excitation wave front during longitudinal conduction in myocardial tissue.