Biosynthesis ofmucin-typeO-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferases, which contain several conserved cysteine resi-dues among the isozymes. We found that a cysteine-specific reagent, p-chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent.