The carboxyl terminal domain of RNA polymerase II has multiple essential roles in transcription initiation, promoter clearance, transcript elongation, and the recruitment of the RNA processing machinery. Specific phosphorylation events are associated with the spatial and temporal coordination of these different activities. The CTD is also modified by beta-O-linked GlcNAc on a subset of RNA Pol II molecules. Using synthetic CTD substrates, we show here that O-GlcNAc and phosphate modification of the CTD are mutually exclusive at the level of the enzymes responsible for their addition. In addition, we show that O-GlcNAc transferase and CTD kinase have different CTD repeat requirements for enzymatic activity. The Km values of the two enzymes for CTD substrates are in a similar range, indicating that neither enzyme has a distinct kinetic advantage. Thus, the in vivo regulation of O-GlcNAc and phosphate modification of the CTD may involve the differential association of these two enzymes with the CTD at specific stages during the transcription cycle. Furthermore, direct competition between OGT and CTD kinase in vivo could generate multiple functionally distinct isoforms of RNA Pol II. Taken together, these results suggest that O-GlcNAc may give rise to additional functional states of RNA Pol II and may coordinate with phosphorylation to regulate class II gene transcription.