In the HERG channel there are at least two binding sites for extracellular protons. One site alters gating, and the other alters conductance. One of these sites shifts activation and inactivation voltage dependence. The only two histidines on the extracellular loops of the channel have been shown to be uninvolved in the pH sensitivity of gating. We hypothesized that a histidine residue located at nearly the midpoint of S5 (H562) could be the pH sensor for gating kinetics. We substituted a glutamine at this point and produced a HERG current with kinetics shifted nearly 20 mV negative and rendered gating pHo-insensitive. This analysis was extended to a HERG construct with all three histidines removed. Although removal of the histidine from S5 in HERG resulted in reduced pH sensitivity of deactivation time constants, this disruption could occur via several mechanisms. Therefore, we tested whether inserting a histidine into the middle of the S5 region of the Kv1.4 channel would confer pH sensitivity. Because of an intrinsic pH sensitivity, we made the S5 mutation in a double construct in which the external pH sensing histidine at position 508 in Kv1.4 was mutated to a glutamine. Introduction of a histidine at position G489 on the S5 voltage sensor of the H508Q mutant channel induced pH dependent gating. Changing pHo from 7.8 to 6.2 caused an approximate −15 mV shift in the threshold for activation of Kv1.4[G489H][H508Q]. The half activation time was pHo dependent for a shift from pH 7.4 to 6.2. The G489H mutation restored the coupling between extracellular pH and N-terminal binding that was disrupted by the H508Q mutation. These studies suggest that an important pH sensitivity of gating is conferred by an S5 cleft space histidine in HERG.