Methionine N(α)-acetyltransferase (M-N(α)AT), which selectively catalyzes the transfer of an acetyl group from acetyl coenzyme A to the α-NH2 group of methionine residues in proteins and peptides, was isolated from Saccharomyces cerevisiae. The enzyme was purified 22 000-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, DE-52 cellulose, CM-52 cellulose, Affi-Gel Blue, gel and hydroxyapatite. The M(r) of the native enzyme was estimated to be 70000 ± 5000 by gel filtration chromatography. The enzyme has a pI near 8.3 as determined by chromatofocusing on Mono P. The enzyme catalyzed the transfer of an acetyl group to a synthetic peptide mimicking the first 24 residues of yeast proteinase A inhibitor 3 (Met-Asn-Thr···) and 3 of its 19 penultimately substituted analogues ([Asp2], [Glu2], and [Gln2]). Based on the estimated molecular weight and amino-acid sequence, The enzyme is different from two other recently identified methionine N(α)-acetyltransferases, NAT2 (Kulkami, M.S. and Sherman, F. (1994) J. Biol. Chem. 269, 13141-13147) and MAK3 (Tercero, J.C. and Wickner, R.B. (1992) J. Biol. Chem. 267, 20277-20281). Among these three enzymes, M-N(α)AT and NAT2 have similar substrate specificity, however, only purified M-N(α)AT, but not recombinant NAT2 gene product, can catalyze the transfer of acetyl group to NH2-terminal methionine residues. The availability of this methionine N(α)-acetyltransferase will advance the understanding of protein co-translational processing.