In the HERG channel there are at least two binding sites for extracellular protons. One site alters gating 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 mid point of S5 could be the pH sensor for gating kinetics if it faced an aqueous cleft space. We constructed a series of point mutations at the location of this histidine and expressed these channels in xenopus oocytes and measured the resulting currents using two electrode or cut-open oocyte clamp. Only one of these mutant channels, glutamine, produced a HERG current with kinetics shifted nearly 20 mV negative and rendered gating pHo-insensitive. This construct has been characterized and extended to a HERG construct with all three histidines removed. This mutation removed the pH dependence of deactivation of the current. There remains a strong component of proton block of the pore, which is not mediated by the histidine residues examined to date. 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 access of a large portion of S5 to the external aqueous phase was a general property of voltage gated potassium channels by attempting to insert this histidine into the middle of the S5 region of the distantly related Kv1.4 channel. Because of an intrinsic pH sensitivity conferred by a histidine at position 508 we made the S5 mutations in a double construct in which the external pH sensing histidine at position 508 was also mutated to a glutamine. Introduction of a histidine at position G489 on the S5 voltage sensor of the H508Q mutant channel showed a pH dependence of gating behavior that was not present in the wild type. Changing extracellular pH from 7.8 to 6.2 causes an approximate 15 mV shift negative in the threshold for activation of Kv1.4[G489H][H508Q]that is not seen in the H508Q mutation alone, the half activation time of Kv1.4[G489H][H508Q] was strongly pHo dependent for a shift from pH 7.4 to 6.2 whereas the half activation time of the H508Q mutant was not. Fast inactivation was also similarly affected. 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 is conferred by an S5 cleft space histidine in HERG and that movement of S5 in Kv1.4 channels can be detected by an introduced histidine at an analogous position.