A novel inhibitor of influenza virus growth in tissue culture was identified and characterized. This compound (BMY-27709) has an IC50 of 3-8 microM for A/WSN/33 virus growth in a multicycle replication assay and is active against all H1 and H2 subtype viruses tested. However, BMY-27709 was found to be inactive against H3 subtype viruses, as well as influenza B/Lee/40 virus. BMY-27709 was also found to act against H1 and H2 viruses early in infection, suggesting that the target for inhibition is the hemagglutinin protein. This was confirmed through the use of reassortant viruses and the isolation of a virus resistant to BMY-27709. The resistant virus isolated contained a phenylalanine to serine change at amino acid 110 of the HA2 subunit. That this single mutation was responsible for the acquisition of resistance to BMY-27709 was proven through reverse genetics, as transfectant virus containing only this change was shown to be resistant to BMY-27709, while the control virus without this mutation remained sensitive. BMY-27709 is able to inhibit virus-induced red blood cell hemolysis, suggesting that it blocks the membrane fusion function of hemagglutinin. These experiments further illustrate that the hemagglutinin protein of influenza virus is a viable target for the discovery and development of small molecule inhibitors of virus growth.