The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II is essential in vivo, and is found in either an unphosphorylated (IIa) or hyperphosphorylated (IIo) form. The Drosophila uninduced hsp70 and hsp26 genes, and the constitutively expressed beta-1 tubulin and Gapdh-2 genes, contain an RNA polymerase II complex which pauses after synthesizing a short transcript. We report here that, using an in vivo ultraviolet crosslinking technique and antibodies directed against the IIa and IIo forms of the CTD, these paused polymerases have an unphosphorylated CTD. For genes containing a 5' paused polymerase, passage of the paused RNA polymerase into an elongationally competent mode in vivo coincides with phosphorylation of the CTD. Also, the level of phosphorylation of the CTD of elongating polymerases is shown not to be related to the level of transcription, but is promoter specific.