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 Neu5Acα2–3Galβ1–4(6-HSO₃)GlcNAcβ (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.