The Mediator complex associates with RNA polymerase II (RNAPII) at least partly via the RNAPII C-terminal repeat domain (CTD). This association greatly stimulates the CTD kinase activity of general transcription factor TFIIH, and subsequent CTD phosphorylation is involved in triggering promoter clearance. Here, highly purified proteins and a protein dissociation assay were used to investigate whether the RNAPII·Mediator complex (holo-RNAPII) can be disrupted by CTD phosphorylation, thereby severing one of the bonds that stabilize promoter-associated initiation complexes. We report that CTD phosphorylation by the serine 5-specific TFIIH complex, or its kinase module TFIIK, is indeed sufficient to dissociate holo-RNAPII. Surprisingly, phosphorylation by the CTD serine 2-specific kinase CTDK1 also results in dissociation. Moreover, the Mediator-induced stimulation of CTD phosphorylation previously reported for TFIIH is also observed with CTDK1 kinase. An unrelated CTD-binding protein, Rsp5, is capable of stimulating this CTD kinase activity as well. These data shed new light on mechanisms that drive the RNAPII transcription cycle and suggest a mechanism for the enhancement of CTD kinase activity by the Mediator complex.