Influenza A viruses of the H3 subtype caused the 1968 Hong Kong pandemic, the hemagglutinin (HA) gene being introduced into humans following a reassortment event with an avian virus. Receptor specificity and serum inhibitor sensitivity of the HA of influenza A viruses are linked to the host species. Human H3 viruses preferentially recognize N-acetyl sialic acid linked to galactose by alpha2,6 linkages (Neu5Acalpha2,6Gal) and are sensitive to serum inhibitors, whereas avian and equine viruses preferentially recognize Neu5Acalpha2,3Gal linkages and are resistant to serum inhibitors. We have examined the receptor specificity and serum inhibitor sensitivity of H3 human influenza A viruses from the time they were introduced into the human population to gain insight into the mechanism of viral molecular evolution and host tropism. All of the viruses were sensitive to neutralization and hemagglutination inhibition by horse serum. Early H3 viruses were resistant to pig and rabbit serum inhibitors. Viruses isolated after 1977 were uniformly sensitive to inhibition by pig and rabbit sera. The recognition of Neu5Acalpha2,3Gal or Neu5Acalpha2,6Gal linkages was not correlated with the serum sensitivity. These data showed that the receptor specificity of HA, measured as inhibitor sensitivity, has changed during replication in humans since its introduction from an avian virus.