In eukaryotes, the Class I actins, β- and γ-actin, match the substrate specificity of NatB and are co-translationally Nt-acetylated by NatB. This acetylated methionine is next cleaved off probably by the enzymatic action of an unidentified aminopeptidase. The newly exposed N-terminus is post-translationally Nt-acetylated by NatH/NAA80. These post-translational steps are specific for animals. The Class II actins, muscle α- and γ-actin, likely undergo co-translational NatA-mediated Nt-acetylation after iMet removal and then follow the same post-translational processing as the Class I actins.