The human hARD1-NATH complex, cotranslationally acetylating the alpha-amino groups of proteins, was recently described. In S. cerevisiae and D. melanogaster this NatA complex contains a third subunit, Nat5p or San, respectively. Based on phylogenetic analyses and database searches, we here describe the human homologue, hNAT5, of these proteins. RT-PCR experiments demonstrated that hNat5 mRNA was expressed in several human cell lines. The candidacy of hNAT5 as a third subunit of the hARD1-NATH complex was investigated using anti-NATH or anti-hARD1 in co-immunoprecipitation experiments followed by Mass Spectrometry analysis of tryptic peptides. Oligopeptides specific for hNAT5 were identified. This verified the expression of endogenous hNAT5 protein in human cells and also identified hNAT5 as a NATH and hARD1 interacting partner. hNAT5 localized to the cytoplasm in accordance with hNAT5-hARD1-NATH complexes playing a role in cotranslational N-alpha-acetylation. Sequence alignment revealed a high degree of similarity of the NAT5 protein between species supporting its conserved role as a part of the complex. The predicted acetyltransferase domain within hNAT5 suggested that this protein, like hARD1, is an enzymatically active component. In summary, we present the first description of the human homologue of Nat5p/San, hNAT5, the third component of the human NatA N-alpha-acetyltransferase complex.