Phosphorylation of RNA polymerase II carboxy-terminal domain (CTD) in hepta-repeats YSPTSPS regulates eukaryotic transcription. Whereas Ser5 is phosphorylated in the initiation phase, Ser2 phosphorylation marks the elongation state. Here we show that the positive transcription elongation factor P-TEFb is a Ser5 CTD kinase that is unable to create Ser2/Ser5 double phosphorylations, while it exhibits fourfold higher activity on a CTD substrate pre-phosphorylated at Ser7 compared with the consensus hepta-repeat or the YSPTSPK variant. Mass spectrometry reveals an equal number of phosphorylations to the number of hepta-repeats provided, yet the mechanism of phosphorylation is distributive despite the repetitive nature of the substrate. Inhibition of P-TEFb activity is mediated by two regions in Hexim1 that act synergistically on Cdk9 and Cyclin T1. HIV-1 Tat/TAR abrogates Hexim1 inhibition to stimulate transcription of viral genes but does not change the substrate specificity. Together, these results provide insight into the multifaceted pattern of CTD phosphorylation.