While the molecular mechanism of membrane fusion by the influenza virus hemagglutinin (HA) protein has been studied extensively in vitro, the role of acid-dependent HA protein activation in virus replication, pathogenesis, and transmission in vivo has not been characterized. To investigate the biological significance of the pH of activation of the HA protein, we compared the properties of four recombinant viruses with altered HA protein acid stability to those of wild-type influenza virus A/chicken/Vietnam/C58/04 (H5N1) in vitro and in mallards. Membrane fusion by wild-type virus was activated at pH 5.9. Wild-type virus had a calculated environmental persistence of 62 days and caused extensive morbidity, mortality, shedding, and transmission in mallards. An N114K mutation that increased the pH of HA activation by 0.5 unit resulted in decreased replication, genetic stability, and environmental stability. Changes of +0.4 and -0.5 unit in the pH of activation by Y23H and K58I mutations, respectively, reduced weight loss, mortality, shedding, and transmission in mallards. An H24Q mutation that decreased the pH of activation by 0.3 unit resulted in weight loss, mortality, clinical symptoms, and shedding similar to those of the wild type. However, the HA-H24(1)Q virus was shed more extensively into drinking water and persisted longer in the environment. The pH of activation of the H5 HA protein plays a key role in the propagation of H5N1 influenza viruses in ducks and may be a novel molecular factor in the ecology of influenza viruses. The data also demonstrate that H5N1 neuraminidase activity increases the pH of activation of the HA protein in vitro.