Natural killer cell (NK) recognition of influenza virus infected cells involves HA binding to sialic acid (SA) on activating NK receptors. SA also acts as a receptor for the binding of influenza virus to its target host cells. The SA binding properties of H3N2 influenza viruses have been observed to change during circulation in humans: recent isolates are unable to agglutinate chicken red blood cells, and show reduced affinity for synthetic glycopolymers representing sialic acid-alpha-2,3-lactose (3´SL-PAA) and sialic acid-alpha-2,6-N-acetyl lactosamine (6´SLN-PAA) carbohydrates. Here, NK lysis of cells infected with human H3N2 influenza viruses isolated between 1969 and 2003 was analysed. Cells infected with recent isolates (1999-2003) were found to be lysed less well than cells infected with older isolates (1969-1996). This change occurred concurrently with the acquisition of two new potential glycosylation site motifs on HA. Deletion of the potential glycosylation site motif at 133-135 in HA1 from a recent isolate partially restored the agglutination phenotype to a recombinant virus, indicating that the HA:SA interaction is inhibited by the glycosylation modification. Deletion of either of the recently acquired potential glycosylation sites on HA led to increased NK lysis of cells infected with recombinant viruses carrying modified HA. These results indicate that alterations in HA glycosylation may affect NK cell recognition of influenza virus-infected cells in addition to virus binding to host cells.