Hemagglutinin (HA) of H3N2/1968 pandemic influenza viruses differed from putative avian precursor by seven amino acid substitutions. Substitutions Q226L and G228S are known to be essential for avian-to-mammalian adaptation. We found that introduction of avian-virus-like amino acids at five other HA positions (62, 81, 92, 144 and 193) of A/Hong Kong/1/1968 virus decreased viral replication in human cells and transmission in pigs. Thus, substitutions at some of these positions facilitated emergence of the pandemic virus.