Acetylation is the most frequently occurring chemical modification of the α-NH2 group of eukaryotic proteins and is catalyzed by an N(α)-acetyltransferase. Recently, a eukaryotic N(α)-acetyltransferase was purified to homogeneity from Saccharomyces cerevisiae, and its substrate specificity was partially characterized (Lee, F.-J.S., Lin L.-W., and Smith, J.A. (1988) J. Biol. Chem. 263, 14948-14955). This article describes the cloning from a yeast λgt11 cDNA library and sequencing of a full length cDNA encoding yeast N(α)-acetyltransferase. DNA blot hybridizations of genomic and chromosomal DNA reveal that the gene (so-called AAA1, amino-terminal, α-amino, acetyltransferase)is present as a single copy located on chromosome IV. The use of this cDNA will allow the molecular details of the role of N(α)-acetylation in the sorting and degradation of eukaryotic proteins to be determined.