Slow conduction and unidirectional conduction block (UCB) are key mechanisms of reentry. Following abrupt changes in heart rate, dynamic changes of conduction velocity (CV) and structurally determined UCB may critically influence arrhythmogenesis. Using patterned cultures of neonatal rat ventricular myocytes grown on microelectrode arrays, we investigated the dynamics of CV in linear strands and the behavior of UCB in tissue expansions following an abrupt decrease in pacing cycle length (CL). Ionic mechanisms underlying rate-dependent conduction changes were investigated using the Pandit-Clark-Giles-Demir model. In linear strands, CV gradually decreased upon a reduction of CL from 500 ms to 230-300 ms. In contrast, at very short CL's (110-220 ms), CV first decreased before increasing again. The simulations suggested that the initial conduction slowing resulted from gradually increasing action potential duration (APD), decreasing diastolic intervals and increasing postrepolarization refractoriness, which impaired I_Na recovery. Only at very short CL's, APD subsequently shortened again due to increasing Na⁺/K⁺ pump current secondary to intracellular Na⁺ accumulation, which caused recovery of CV. Across tissue expansions, the degree of UCB gradually increased at CL's of 250-390 ms, while, at CL's of 180-240 ms, it first increased and subsequently decreased. In the simulations, reduction of inward currents caused by increasing [Na⁺]_i and [Ca²⁺]_i contributed to UCB progression, which was reversed by increasing Na⁺/K⁺ pump activity. In conclusion, CV and UCB follow intricate dynamics upon an abrupt decrease in CL, which are determined by the interplay between I_Na recovery, postrepolarization refractoriness, APD changes, ion accumulation and Na⁺/K⁺ pump function.