In order to establish the sequence dependence of RimJ-mediated protein N-terminal acetylation in E. coli, the Z-domain variants differing by the second or third amino acid residue were expressed and analyzed by mass spectrometry. Only subsequent to the initiating methionine residue cleavage, the RimJ-catalyzed N-terminal acetylation mainly occurred at the N-terminal serine and threonine residues and was significantly enhanced by hydrophobic or negatively charged residues in the penultimate position.