N(alpha)-terminal acetylation occurs in the yeast Saccharomyces cerevisiae by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain Ard1p, Nat3p and Mak3p catalytic subunits, respectively. The N-terminal sequences required for N-terminal acetylation, i.e. the NatA, NatB, and NatC substrates, were evaluated by considering over 450 yeast proteins previously examined in numerous studies, and were compared to the N-terminal sequences of more than 300 acetylated mammalian proteins. In addition, acetylated sequences of eukaryotic proteins were compared to the N termini of 810 eubacterial and 175 archaeal proteins, which are rarely acetylated. Protein orthologs of Ard1p, Nat3p and Mak3p were identified with the eukaryotic genomes of the sequences of model organisms, including Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Mus musculus and Homo sapiens. Those and other putative acetyltransferases were assigned by phylogenetic analysis to the following six protein families: Ard1p; Nat3p; Mak3p; CAM; BAA; and Nat5p. The first three families correspond to the catalytic subunits of three major yeast NATs; these orthologous proteins were identified in eukaryotes, but not in prokaryotes; the CAM family include mammalian orthologs of the recently described Camello1 and Camello2 proteins whose substrates are unknown; the BAA family comprise bacterial and archaeal putative acetyltransferases whose biochemical activity have not been characterized; and the new Nat5p family assignment was on the basis of putative yeast NAT, Nat5p (YOR253W). Overall patterns of N-terminal acetylated proteins and the orthologous genes possibly encoding NATs suggest that yeast and higher eukaryotes have the same systems for N-terminal acetylation.