Growth of clinical specimens of influenza viruses in eggs can result in the selection of antigenic variants distinct from corresponding viruses grown in mammalian tissue culture. To evaluate the contribution of host cell selection on the antigenic diversity of human influenza isolates, as seen in annual surveillance studies, viruses grown in embryonated eggs were compared by antigenic and genetic analyses with their mammalian tissue culture-grown counter parts. Clinical specimens were gathered from around the world from late 1987 to 1990 and the antigenicity of isolated viruses was assessed by hemagglutination-inhibition assays using immune ferret sera as is currently performed for routine surveillance and the selection of vaccine strains. In addition, viruses were assessed using a panel of anti-H3 HA monoclonal antibodies. The extent of antigenic variation exhibited by the egg-grown strains was far greater than the relative antigenic homogeneity of the tissue culture-grown viruses. Nucleotide sequence analysis of HA1 gene PCR products of 28 MDCK cell and egg derived pairs allowed identification of amino acid substitutions responsible for the antigenic differences observed and the adaptation to growth in eggs Among these substitutions was a change at amino acid position 186 of HA1 (Ser in tissue culture viruses and Ile in egg-grown viruses) which was observed at relatively high frequency. Egg- and MDCK-grown pairs with this single amino acid difference were classified into distinct antigenic groups by ferret sera raised to WHO reference viruses. Given the additional antigenic diversity observed among egg-grown strains, considerable care should be taken in the selection of reference and vaccine strains grown in eggs. Rapid sequence comparisons of MDCK- and egg-grown viruses allow identification of variants arising through egg selection and will prove to be a useful adjunct to antigenic surveillance for the selection of reference and vaccine strains. © 1993 Academic Press, Inc.