The major site of acetylation of glyceraldehyde-3-phosphate dehydrogenase [ d -glyceraldehyde 3-phosphate; NAD oxidoreductase (phosphorylating) EC 1.2.1.12] by p -nitrophenylacetate is the sulfhydryl group of the cysteine residue in the peptide, Ala-Ser-CySAc, derived from the acetylated enzyme by pepsin digestion. If all of the peptides analyzed come from the same section of the peptide chain of the protein, then the sequence about this site of acetylation appears to be Ala-Ser-CySAc-(Asp, Thr, Thr). The relationship of these results to the peptide Ser-CM-CyS-(AspNH 2 , Thr) obtained by Harris and Park from pepsin digests of the enzyme after reaction with iodoacetate is discussed. These data do not provide support for the earlier concept that glutathione or a glutathione-like portion of the peptide chain was the site of similar reactions.