Aspirin (acetylsalicyclic acid) inhibits prostaglandin synthesis by acetylating a single internal serine residue of the initial enzyme in the biosynthetic pathway, prostaglandin synthetase. The region of the enzyme that is modified by aspirin was isolated, and its amino acid sequence was determined. Sheep vesicular gland [acetyl-3H]prostaglandin synthetase was purified following treatment with [acetyl-3H]aspirin and digested with pepsin. An acetyl-3H-labeled peptic peptide of .apprx. 25 residues was isolated by high-pressure liquid chromatography, and its amino acid sequence was determined. The acetylated serine residue was located at position 8 in this sequence. The current study marks this polypeptide sequence as a region related to an active site of the enzyme.