Hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change at acidic pH, mediating viral fusion with the host endosomal membrane. To unravel the molecular basis of the pH-dependent stability of HA, we demonstrate by mutagenesis of the prototype HA of virus strain X31 (H3 subtype) that salt bridges, especially a tetrad salt bridge within the monomers, are crucial for folding and stability of the trimeric ectodomain. This complex (tetrad) salt bridge is highly conserved among influenza virus subtypes. Introducing additional sites of electrostatic attraction between monomers in the distal region enhanced the stability of ectodomain at low pH mimicking the natural variant H2 subtype. We propose that distinct salt bridges in the distal domain may contribute to the enhanced stability of HA of natural virus variants. This hypothesis may provide clues to understanding adaptations of virus strains (for example, avian influenza viruses) in order to preserve stability of the protein in the host-specific environment.--Rachakonda, P. S., Veit, M., Korte, T., Ludwig, K., Böttcher, C., Huang, Q., Schmidt, M. F. G., Herrmann, A. The relevance of salt bridges for the stability of the influenza virus hemagglutinin.