Influenza A virus NS1 protein is an important regulatory factor with multiple functions and contributes greatly to viral pathogenesis. In the present study, transcription-activating potential of NS1 from different influenza A virus subtypes was examined in yeast two-hybrid system. The bait vectors containing different NS1 genes, along with an empty prey vector, were transformed into yeast AH109(for growth assay on QDO plate and alpha-galactosidase assay), and Y187(for beta-galactosidase assay). AH109 transformants with NS1 gene from H1N1, H5N1, and H9N2 viruses grew vigorously on the QDO plate and secreted high level of alpha-galactosidase. Also, Y187 bearing the above NS1 genes exhibited enhanced beta-galactosidase activity. Nevertheless, H3N2-NS1-transformed AH109 and Y187 yeasts did not grow on QDO plate and secrete beta-galactosidase, respectively. These findings denote the remarkable variation in NS1 proteins from different influenza A virus subtypes on the transcription-stimulating capability in yeast.