Antigenic variation among influenza B viruses is different from that of influenza A in several ways. Antigenic shift has not been observed, distinct antigenic variants of influenza B cocirculate, and antigenically similar viruses have been isolated many years apart. To study the mechanism of antigenic drift in influenza B viruses, monoclonal antibodies were used to select antigenic variants of B/Hong Kong/8/73 virus hemagglutinin (HA). Analyses of the nucleotide sequences of the HA gene of B/Hong Kong/8/73 and the eight variants identified specific regions of the influenza B HA molecule involved in antigenicity, and enabled antigenic mapping data to be correlated with the structure of the protein. The altered amino acids in the variants, when compared to the HA of A/Aichi/2/68, were found in two of the four antigenic regions previously identified for type A viruses. In addition, four of the eight variants showed multiple nucleotide changes some of which gave rise to double amino acid changes. In addition, in the present study monoclonal antibodies which belong to the same antigenic group recognize amino acid changes in regions corresponding to antigenic sites A and B of the H3 HA. These results are in contrast to those obtained with HA variants of A/Memphis/1/71 virus. In the influenza A studies only single amino acid changes were found and these correlated well with the three-dimensional structure as determined by D. C. Wiley, I. A. Wilson, and J. J. Skehel, (1981, Nature (London) 289, 366-373); monoclonal antibodies which recognized one region did not recognize any of the other antigenic sites. Our results suggest that although the basic three-dimensional structure of the influenza B HA may be similar to that of A viruses, the B HA molecule may be folded in a more compact manner so that antigenic sites A and B are in closer proximity to each other than in the H3 structure.