The largest subunit of mammalian RNA polymerase II (RNAP II) contains at its carboxyl terminus an unusual domain consisting of 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain, designated the COOH-terminal domain (CTD), is essential for viability and is extensively phosphorylated during the transition from preinitiation complex assembly to elongation (1). Indeed, phosphorylation of the CTD may play an important regulatory role in this transition. We show here that the CTD is also modified by a novel form of protein glycosylation, O-GlcNAc. This modification has been found on numerous transcription factors and other nuclear and cytosolic proteins (2). Glycopeptides obtained by proteolytic digestion of the CTD were purified by reverse-phase high performance liquid chromatography and sequenced. Results from such experiments suggest that glycosylation occurs at multiple sites throughout the CTD, similar to the phosphorylation of this domain. The carbohydrate, however, is not detectable on the phosphorylated form of the enzyme. This observation is consistent with the idea that phosphorylation and glycosylation are mutually exclusive modifications. The CTD of RNAP II, therefore, appears to exist in three distinct conformational states: unmodified, phosphorylated, and glycosylated. The differential modification of the CTD may play an important role in the regulated expression of genes transcribed by RNA polymerase II.