Incubation of purified prostaglandin endoperoxide synthetase from sheep vesicular glands with aspirin results in a covalent binding of the acetyl group of acetylsalicylic acid to the protein. During this acetylation, the cyclooxygenase activity is lost, but not the peroxidase activity. The reaction is completed when almost one acetyl group is bound per polypeptide chain (Mr = 68 000). After proteolysis of [3H]acetyl-protein with pronase, radioactive N-acetylserine was obtained. Originally, however, the hydroxyl group of an internal serine residue in the chain is acetylated. The formation of N-acetylserine can be explained by a rapid O leads to N acetyl shift as soon as the NH2 group of serine is liberated. A radioactive dipeptide was isolated from a thermolysin digest of the [3H]acetyl-enzyme containing phenylalanine and serine, phenylalanine being its N-terminal amino acid. Automatic Edman degradation of native and acetylated enzyme showed that only one polypeptide sequence was present: Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-X-X-Tyr-. The N-terminal sequence has an apolar character.