Influenza viruses gain entry into host cells by binding to cellular receptors and promoting the fusion of the viral envelope with the host cell membrane. The fusion peptide of influenza hemagglutinin (HA) is crucial for fusion. To examine the structural and functional roles of amino acids E11 and G8 of the H5 HA fusion peptide, a series of fusion mutants was generated. We determined the effect of each mutation on fusion activity and infection of rescued recombinant virus by polykaryon formation, cell-cell fusion assay, HA pseudovirus transduction and reverse genetics. Our findings indicate that E11V and E11A mutants dramatically inhibit fusion and that at position 11 a polar residue such as glutamic acid or serine may be desirable for preserving the fusion activity. More interestingly, one mutation (G8E) raised the threshold pH of polykaryon formation. Our results suggest that G8 as well as E11 play an important functional and structural role in membrane fusion and that the polarity of E11 is crucial for fusion activity. Finally, we developed an assay based on a reporter gene plus pseudotyped virus that could sensitively detect fusion activity.