Anisotropic impulse conduction was studied in neonatal rat heart cell monolayers produced by culturing cells on a growth-directing substrate of collagen. Monolayers consisting of parallel-oriented cells without visible intercellular clefts were selected for experiments; cell lengths and widths were 65.8±12.5 and 12.2±3.2 μm (n=49), respectively. Action potential upstrokes were measured by using 12 photodiodes selected within a 10x10 diode array and a voltage-sensitive dye (RH-237). The size of the area sensed by a single diode was 14x14 μm. High-density multiple recordings (resolution, up to 15 μm) demonstrated the variability of local activation delays and of the maximal rate of rise of the action potential upstroke (V(max)), which are presumably related to the microscopic cellular architecture. Mean macroscopic conduction velocities measured over distances of 135 μm were 34.6±4.5 and 19.0±4.3 cm/s (mean±SD, n=13, P<.0001) in longitudinal and transverse directions, respectively. The anisotropic velocity ratio was 1.89±0.38 (n=13). Mean V(max) was not significantly different in two directions (122.0±17.4 V/s longitudinally versus 125.2±15.6 V/s transversely, n=13, P=NS). In conclusion, we developed an anisotropic cell culture model suitable for studying impulse conduction with cellular resolution. The anisotropic velocity ratio was close to values measured in vivo. By contrast, V(max) was not dependent on the direction of propagation.