Objective: Unidirectional conduction block (UCB) and reentry may occur as a consequence of an abrupt tissue expansion and a related change in the electrical load. The aim of this study was to evaluate critical dimensions of the tissue necessary for establishing UCB in heart cell culture. Methods: Neonatal rat heart cell cultures with cell strands of variable width emerging into a large cell area were grown using a technique of patterned cell growth. Action potential upstrokes were measured using a voltage sensitive dye (RH-237) and a linear array of 10 photodiodes with a 15 μm resolution. A mathematical model was used to relate action potential wave shapes to underlying ionic currents. Results: UCB (block of a single impulse in anterograde direction - from a strand to a large area - and conduction in the retrograde direction) occurred in narrow cell strands with a width of 15(SD 4) μm (1-2 cells in width, n = 7) and there was no conduction block in strands with a width of 31(8) μm (n = 9, P < 0.001) or larger. The analysis of action potential waveshapes indicated that conduction block was either due to geometrical expansion alone (n = 5) or to additional local depression of conduction (n = 2). In wide strands, action potential upstrokes during anterograde conduction were characterised by multiple rising phases. Mathematical modelling showed that two rising phases were caused by electronic current flow, whereas local ionic current did not coincide with the rising portions of the upstrokes. Conclusions (1) High resolution optical mapping shows multiphasic action potential upstrokes at the region of abrupt expansion. At the site of the maximum decrement in conduction, these peaks were largely determined by the electrotonus and not by the local ionic current. (2) Unidirectional conduction block occurred in strands with a width of 15(4) μm (1-2 cells).