Influenza virus hemagglutinin (HA) mediates viral entry into cells by a low pH-induced membrane fusion event in endosomes. A number of structural changes occur throughout the length of HA at the pH of fusion. To probe their significance and their necessity for fusion activity, we have prepared a site-directed mutant HA containing novel intersubunit disulfide bonds designed to cross-link covalently the membrane-distal domains of the trimer. These mutations inhibited the low pH-induced conformational changes and prevented HA-mediated membrane fusion; conditions that reduced the novel disulfide bonds restored membrane fusion activity. We conclude that structural rearrangements in the membrane distal region of the HA are required for membrane fusion activity.