Mammalian cells contain two forms of RNA polymerase II, designated IIO and IIA, that differ in the extent of phosphorylation within the C-terminal domain of their largest subunit. Phosphorylation of this domain, which results in the conversion of RNA polymerase IIA to IIO, may play an important role in the transition from the initiation to the elongation phase of transcription. A third form of the enzyme, RNA polymerase IIB, is found in vitro and lacks the repetitive C-terminal domain. Purified calf thymus RNA polymerase IIA was labeled selectively with casein kinase II in the presence of [gamma-32P]ATP and used as substrate for the identification and partial purification of factors that catalyze the conversion of RNA polymerase IIA to IIO. HeLa cell S-100 transcription extracts contain such an activity that cofractionates with factors essential for promoter-dependent transcription through heparin-Sepharose, DEAE-5PW, and DE52 chromatography. The activity is dependent on either ATP, GTP, or dATP, requires a hydrolyzable beta,gamma-phosphoanhydride bond, and cannot utilize pyrimidine nucleoside triphosphates. This observation supports the idea that the conversion activity is a protein kinase. Transcription of the major late promoter of adenovirus-2 was carried out in the presence of a reconstituted transcription extract containing purified RNA polymerases IIO, IIA, or IIB, and the nature of the elongating enzyme was determined by photoaffinity labeling. When the reaction was initiated with RNA polymerase IIO or IIB, nascent transcripts were found cross-linked to subunit IIo or IIb, respectively. However, when the reaction was initiated with RNA polymerase IIA, nascent transcripts were cross-linked to subunit IIo. Consequently, phosphorylation of the C-terminal domain of subunit IIa must have occurred prior to elongation. The copurification of RNA polymerase IIA to IIO conversion activity with factors essential for promoter-dependent transcription and the observation that RNA polymerase II containing an unphosphorylated C-terminal domain is phosphorylated prior to elongation suggest that protein kinases that phosphorylate the C-terminal domain of subunit IIa may play an essential role in transcription.