AbstractN‐terminal acetylation is one of the most common modifications, occurring on the vast majority of eukaryotic proteins. Saccharomyces cerevisiae contains three major NATs, designated NatA, NatB, and NatC, with each having catalytic subunits Ard1p, Nat3p, and Mak3p, respectively. Gautschi et al. (Gautschi et al. [2003] Mol Cell Biol 23: 7403) previously demonstrated with peptide crosslinking experiments that NatA is bound to ribosomes. In our studies, biochemical fractionation in linear sucrose density gradients revealed that all of the NATs are associated with mono‐ and polyribosome fractions. However only a minor portion of Nat3p colocalized with the polyribosomes. Disruption of the polyribosomes did not cause dissociation of the NATs from ribosomal subparticles. The NAT auxiliary subunits, Nat1p and Mdm20p, apparently are required for efficient binding of the corresponding catalytic subunits to the ribosomes. Deletions of the genes corresponding to auxiliary subunits significantly diminish the protein levels of the catalytic subunits, especially Nat3p, while deletions of the catalytic subunits produced less effect on the stability of Nat1p and Mdm20p. Also two ribosomal proteins, Rpl25p and Rpl35p, were identified in a TAP‐affinity purified NatA sample. Moreover, Ard1p copurifies with Rpl35p‐TAP. We suggest that these two ribosomal proteins, which are in close proximity to the ribosomal exit tunnel, may play a role in NatA attachment to the ribosome. J. Cell. Biochem. 103: 492–508, 2008. © 2007 Wiley‐Liss, Inc.