Connexin40 (Cx40) is abundantly expressed in the atrial myocardium, ventricular conduction system, and vascular endothelial and smooth muscle cells of the mammalian cardiovascular system. Rapid conduction through cardiac tissues depends on the electrotonic transfer of the action potential between neighboring cells. To determine if the transjunctional voltages elicited by an action potential can modulate the conductance of Cx40 gap junctions, simulated myocardial action potentials were applied as voltage clamp waveforms to Cx40 gap junctions expressed in mouse neuro2A (N2A) cells. Junctional currents resembled the action potential morphology, but declined by >50% from peak to near constant plateau values. The kinetics of Cx40 voltage gating were examined at peak voltages 100 mV and the decay time constants were found to change e-fold per 17.6 mV for V_j > ±40 mV. Junctional conductance recovered during phase 3 repolarization and early diastole to initial values. These phasic changes in junctional conductance were due to the rapid decay kinetics, increasing to tens of milliseconds at peak transjunctional voltages (V_j) of 130 mV, and the increase in the steady state conductance curve as V_j returns towards 0 mV. The time-dependent conductance curves for Cx40 were modeled with one inactivation and two recovery V_j-dependent components. There was a temporal correlation between the development of conduction delay or block and the inactivation phase of junctional conductance. Likewise, the recovery of junctional conductance was coincident with the recovery from refractoriness, suggesting that gap junctions may play a role in the genesis and propagation of cardiac arrhythmias.