N(α)-Acetylation is a major co-translational modification occurring at the α-NH2 group of eukaryotic cytosolic proteins. In order to understand better the specificity of N(α)-acetyltransferase, we used the purified enzyme from yeast (Lee, F.-J.S., Lin, L.-W., and Smith J.A. (1988) J. Biol. Chem. 263, 14948-14955) and synthetic peptides mimicking the NH2 terminus of yeast and human proteins. Alcohol dehydrogenase I-(1-24) and 8 of the 19 synthetic analogues with substitutions at the NH2-terminal residue were N(α)-acetylated with varying efficiency. Penultimate amino acid substitutions, except for proline, had little influence on N(α)-acetylation. Substitution of sequences from N(α)-acetylated proteins into the yeast sequences which cannot be N(α)-acetylated demonstrated that not only the first 3 NH2-terminal residues but also more carboxyl-terminal residues were important for determining the specificity of N(α)-acetyltransferase. Two other peptides mimicking yeast mitochondrial cytochrome c oxidase (subunit VI) and ATPase inhibitor, which are naturally non-acetylated, were efficiently acetylated. In addition, recombinant human alcohol dehydrogenase I and basic fibroblast growth factor, which are naturally N(α)-acetylated, were not acetylated post-translationally.