We mapped the hemagglutinin (HA) antigenic epitopes of a highly pathogenic H5N1 influenza virus on the three-dimensional HA structure by characterizing escape mutants of a recombinant virus containing A/Vietnam/1203/04 (H5N1) DeltaHA and neuraminidase genes in the genetic background of A/Puerto Rico/8/34 (H1N1) virus. The mutants were selected with a panel of 8 anti-HA monoclonal antibodies (MAbs), 7 to A/Vietnam/1203/04 (H5N1) virus and one to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and mutants´ HA genes were sequenced. The amino acid changes suggested 3 MAbs groups: 4 MAbs reacted with the complex epitope comprising parts of the antigenic site B of H3 HA and site Sa of H1 HA, 2 MAbs reacted with the epitope corresponding to the antigenic site A in H3 HA, and 2 Mabs displayed unusual behavior: each recognized amino acid changes at two widely separate antigenic sites. Five changes were detected in amino acid residues not previously reported as changed in H5 escape mutants, and 4 others had substitutions not previously described. The HA antigenic structure differs substantially between A/Vietnam/1203/04 (H5N1) virus and the low-pathogenic A/Mallard/Pennsylvania/10218/84 (H5N2) virus we previously characterized (Kaverin et al., 2002, J. Gen. Virol. 83:2497-2505). The HI reactions of the MAbs with recent highly pathogenic H5N1 viruses were consistent with the antigenic-site amino acid changes but not with clades and subclades based on H5 phylogenetic analysis. These results provide information on the recognition sites of the MAbs widely used to study H5N1 viruses and demonstrate involvement of the HA antigenic sites in the evolution of highly pathogenic H5N1 viruses, and can be critical for characterizing pathogenesis and vaccine design.