The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pKa matches the pH of late endosomes (pKa～6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines by different amino acids and analyzing the effect of these mutations on conformational change and fusion we found that His184, but not His110, plays an essential role for the pH dependence of conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central of a conserved interaction network possibly regulating the pH dependence of conformational change via its pKa. As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in vicinity of histidine may affect its pKa. HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly as well as the low pathogenic H5 HA we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pKa modulating effect by neighboring residues.