The influence of the host cell on the selection of antigenic variants of influenza A H3N2 viruses and the relevance of host cell selection to the induction of immunity by these viruses have been investigated. Influenza viruses were isolated from human clinical samples during a single epidemic, were passaged in mammalian Madin-Darby Canine Kidney (MDCK) cells or in embryonated hens eggs, and were tested for antigenic variability in the hemagglutinin (HA) molecule with a panel of monoclonal antibodies. In many cases, the HA of virus cultivated in eggs was antigenically distinct from the HA of virus from the same individual grown in mammalian cells. Viruses recovered from different individuals were antigenically similar to each other when grown in mammalian cell lines yet were antigenically heterogeneous when cultivated in eggs. The HA genes of viruses isolated from different individuals during the epidemic were shown, by sequence analysis, to differ from each other by five or six amino acid residues. Sequence analyses of the HA genes of MDCK cell-grown and egg-grown virus obtained from the same individual demonstrated that the molecular changes between antigenically distinct HAs of MDCK cell- and egg-grown A/Mem/12/85 virus involved a single amino acid substitution at residue 156 in HA1, which lies at the tip of the HA molecule and immediately adjacent to the receptor-binding site. However, the amino acid sequences of HAs from MDCK-grown and egg-grown viruses (A/Mem/2/85) isolated from a second individual were identical although these viruses exhibited antigenic differences when examined with anti-HA monoclonal antibodies. Therefore, single amino acid changes in the HA molecule may not be the sole cause of antigenic changes in the HA observed between pairs of MDCK cell-grown and egg-grown viruses and genes other than that encoding the HA may contribute to the host cell-mediated antigenic variation of these viruses. Nevertheless, antigenic differences between viruses grown in eggs and MDCK cells did not influence their ability to protect, since ferrets infected with either live egg-grown or MDCK-grown virus were protected equally well from challenge with virus grown in either host cell type.