The human ether-a-go-go related gene (hERG) potassium channel is a key component of the repolarization of the ventricular action potential and a modulator of neuronal excitability. The N-terminal regions of hERG are known to contribute to the modulation of inactivation and deactivation gating; however, the specific contributions of N-terminal domains and the mechanism of modulation have not been determined. The data presented here demonstrate a key modulatory role by an N-terminal region through interaction with an intracellular C-terminal domain. Using a genetically encoded ether-a-go-go/Per-Arnt-Sim (eag/PAS) domain fragment and FRET spectroscopy, we showed that the eag/PAS domain was the primary determinant of deactivation gating and was able to form a stable interaction with N-truncated channels and to compensate for eag/PAS domain mutations in full length channels. We hypothesized that the C-terminal cyclic nucleotide binding domain (CNBD) was a site of interaction of the eag/PAS domain. CNBD-deleted channels were shown to have abnormal deactivation gating, similar to that seen with N-terminal region-deleted channels. A protein interaction experiment showed direct binding between the eag/PAS domain and a C-linker/CNBD protein. This interaction was demonstrated to occur between adjacent subunits of the hERG channel. To further investigate the role of N-terminal domains in gating, we probed N-terminal region deleted channels with eag/PAS and proximal domain fragments, and determined that the eag/PAS domain is also a key regulator of hERG rectification and inactivation properties; whereas, the proximal domain is involved only in steady-state activation gating. Together, our data indicate that the eag/PAS domain is a key modulator of hERG gating that functions to modulate gating by a direct interaction with the CNBD.