Influenza virus assembles and buds at the infected-cell plasma membrane. This involves extrusion of the plasma membrane followed by scission of the bud, resulting in severing the nascent virion from its former host. The influenza virus M2 ion channel protein contains in its cytoplasmic tail a membrane proximal amphipathic helix that facilitates the scission process and is also required for filamentous particle formation. Mutation of five conserved hydrophobic residues to alanine within the amphipathic helix (5PM) reduced scission and also filament formation, whereas single mutations had no apparent phenotype. Here we show that any two of these five residues mutated together to alanine result in virus debilitated for growth and filament formation in a manner similar to 5PM. Growth kinetics of the M2 mutants are approximately two logs lower than wild type and plaque diameter was significantly reduced. When the 5PM mutations and a representative double mutant (I51A-Y52A) were introduced into A/WSN/33 M2, a strain that produces spherical particles, similar debilitation in viral growth occurred. Electron microscopy showed that with the 5PM and I51A-Y52A A/Udorn/72 and WSN viruses, scission failed and emerging virus particles exhibited a "beads on a string" morphology. The major spike glycoprotein hemagglutinin is localized within lipid rafts in virus-infected cells, whereas M2 is associated at the periphery of rafts. Mutant M2s were more widely dispersed and their abundance at the raft periphery was reduced, suggesting the M2 amphipathic helix is required for proper localization in the host membrane and this has implications for budding and scission.