AbstractNatA is the major N‐terminal acetyltransferase of the yeast Saccharomyces cerevisiae. In this study, we took advantage of our recent data on N‐terminal acetylation of proteins of the yeast protein map to update the list of proteins with known NatA‐dependent acetylation status. Furthermore, using the information available on the acetylation status of 100 novel proteins, we re‐examined the rules for acetylation by NatA. The results refine our previous knowledge on NatA substrate specificity depending on the N‐terminal and penultimate residues. In particular, we found that the acetylation frequencies of Ser‐, Thr‐ and Ala‐, the three residues most often acetylated by NatA, are higher than previously reported. In addition, comparison of the N‐terminal region of acetylated and non‐acetylated proteins revealed differences in amino acid composition that extend over the 25 first amino acid residues: acetylated proteins are characterized by a higher frequency of glutamate and glutamine and a lower frequency of lysine, arginine and histidine. We suggest that the particularities in amino acid composition of the N‐terminal region of acetylated proteins facilitate its interaction with the Nat1p subunit of NatA and its guidance to the catalytic subunit Ard1p. Copyright © 2008 John Wiley & Sons, Ltd.