We have constructed an operational antigenic map of the hemagglutinin of influenza virus A/PR/8/34, which indicates the presence of five immunodominant antigenic regions exhibiting various degrees of operational linkage. These sites have been located by the identification of changed amino acid residues in mutant viruses that are antigenically altered at each site. Comparison of the antigenic features with the three-dimensional structure of the H3 subtype hemagglutinin shows that the antigenic sites correspond to four topographically distinct regions of the surface of the protein. One of the sites is formed when two regions that are widely separated in the hemagglutinin monomer associate in the assembled trimer. The location of the sites relative to those proposed for the H3 subtype hemagglutinin suggests that carbohydrate modulates the antigenicity of specific regions of the hemagglutinin.