Influenza A viruses (H3N2) isolated in MDCK cells after 1992/1993 influenza season have changed in that these viruses agglutinate human red blood cells (HRBC) but not chicken RBC (CRBC) (Ch- virus). The yield of Ch- virus in MDCK cells was comparable with that of the virus that could agglutinate CRBC (Ch+ virus); however, binding assays revealed reduced binding of the Ch- virus to MDCK cells. Replacement of the HA gene of A/Aichi/2/68 (Ch+) with that of A/Aichi/30/97 (Ch-) led to a reduction in binding of the resultant virus to MDCK cells. Ch- viruses, however, efficiently bound to derivative MDCK cells resialylated with SAα2,6Galβ1,4GlcNAc, indicating that HA of Ch- viruses distinguished the sialyloligosaccharides on native MDCK cells from those on resialylated MDCK cells. Ch- viruses exhibited limited sensitivity to NA-specific inhibitor in plaque reduction assays using MDCK cells, despite the fact that NA activity of the Ch- viruses was susceptible to the drug. These findings showed that change in the receptor-binding specificity of Ch- viruses affected the NA-dependent virus released from MDCK cells.