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 mu m (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 14 x 14 mu m High-density multiple recordings (resolution, up to 15 mu m) 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 mu m 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.