Abstract: The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, acts as a binding platform for various mRNA processing and histone-modifying enzymes that act co-transcriptionally. These factors are targeted to specific phosphorylation states of the CTD that predominate at different stages of transcription. Within the repeating sequence YSPTSPS, serines 2 and 5 are major phosphorylation sites, but serine 7 phosphorylation was recently discovered in mammalian cells. Here we show that CTD serine 7 is also phosphorylated in yeast and that Ser7P ChIP patterns resemble those of Ser5P. The basal factor TFIIH can phosphorylate Ser7 in vitro and is necessary for Ser7P in vivo. Interestingly, deletion of the CTD Ser5P phosphatase Rtr1 leads to an increase in Ser5P but not Ser7P.