Influenza virus assembly and budding occur in the ´budozone´, a coalesced raft domain in the plasma membrane. The viral transmembrane protein M2 is implicated in virus particle scission, the ultimate step in virus budding, probably by wedge-like insertion of an amphiphilic helix into the membrane. In order to do so, M2 is hypothesised to be targeted to the edge of the budozone, mediated by acylation and cholesterol binding. We have recently shown that acylation and cholesterol binding affect membrane association of M2´s cytoplasmic tail and targeting of the protein to coalesced rafts. In this study, we tested whether the combined removal of the acylation site (C50) and the cholesterol-binding CRAC motifs (key residues Y52, Y57) in M2´s amphiphilic helix influence virus formation.We generated recombinant influenza viruses in the WSN background with mutations in one or both of these features. All these viruses showed, in comparison with the wildtype, very similar growth kinetics in various cell types. Wildtype and mutant viruses differed in their relative M2 content, but not regarding the major structural proteins. The morphology of the viruses was not affected by mutating M2. Moreover, wildtype and mutant viruses showed comparable competitive fitness in infected cells. Lastly, a global comparison of M2 sequences revealed that there are natural virus strains with M2 devoid of both lipid-association motifs. Taken together, acylation and cholesterol-binding motifs in M2 are not crucial for replication of influenza virus in cell culture, indicating that also other factors can target M2 to the budding site.