Receptor specificity of influenza A/H5 viruses including human 2003-04 isolates was studied. All but two isolates preserved high affinity to Sia2-3Gal (avian-like) receptors. However, two isolates (February, 2003, Hong Kong) demonstrated decreased affinity to Sia2-3Gal and moderate affinity to a Sia2-6Gal (human-like) receptors. These two viruses had a unique Ser227-Asn change in the hemagglutinin molecule. Thus, a single amino acid substitution can significantly alter receptor specificity of avian H5N1 viruses, providing them with an ability to bind to receptors optimal for human influenza viruses. Asian 2003-04 H5 isolates from chickens and humans demonstrated highest affinity to the sulfated trisaccharide Neu5Acalpha2-3Galbeta1-4(6-HSO3)GlcNAcbeta (Su-3'SLN) receptor but, in contrast to 1997 isolates, had increased affinity to fucosylated Su-3'SLN. American poultry H5 viruses also had increased affinity to Su-3'SLN. These data demonstrate that the genetic evolution of avian influenza A(H5N1) viruses is accompanied during adaptation to poultry by the evolution of their receptor specificity.