The receptor specificity of 56 H2 and H3 influenza virus isolates from various animal species has been determined to test the relevance of receptor specificity to the ecology of influenza virus. The results show that the receptor specificity of both H2 and H3 isolates evaluated for sialic acid linkage specificity and inhibition of hemagglutination by horse serum correlates with the species of origin, as postulated earlier for H3 strains based on a limited survey of five human, three avian, and one equine strain. Elucidation of the amino acid sequence of several human H2 receptor variants and analysis of known sequences of H2 and H3 isolates revealed that receptor specificity varies in association with an amino acid change at residues 228 in addition to the change at residue 226 previously documented to affect receptor specificity of H3 but not H1 isolates. Residues 226 and 228 are leucine and serine in human isolates, which preferentially bind sialic acid alpha 2,6-galactose beta 1,4-N-acetyl glucosamine (SA alpha 2,6Gal), and glutamine and glycine in avian and equine isolates, which exhibit specificity for sialic acid alpha-2,3-galactose beta-1,3-N-acetyl galactosamine (SA alpha 2,3Gal). The results demonstrate that the correlation of receptor specificity and species of origin is maintained across both H2 and H3 influenza virus serotypes and provide compelling evidence that influenza virus hosts exert selective pressure to maintain the receptor specificity characteristics of strains isolated from that species.