Abstract: Eukaryotic RNA polymerase II (RNAPII) is a 12-subunit enzyme that is responsible for the transcription of messenger RNA. Two of the subunits of RNA polymerase II, Rpb4 and Rpb7, have been shown to dissociate from the enzyme under a number of specific laboratory conditions. However, a biological context for the dissociation of Rpb4 and Rpb7 has not been identified. We have found that Rpb4/7 dissociate from RNAPII upon interaction with specific transcriptional elongation-associated proteins that are recruited to the hyperphosphorylated form of the C-terminal domain. However, the dissociation of Rpb4/7 is likely short lived because a significant level of free Rpb4/7 was not detected by quantitative proteomic analyses. In addition, we have found that RNAPII that is isolated through Rpb7 is depleted in serine 2 C-terminal domain phosphorylation. In contrast to previous reports, these data indicate that Rpb4/7 are dispensable during specific stages of transcriptional elongation in Saccharomyces cerevisiae.