A kinase activity specific for the C-terminal repeat domain (CTD) of RNA polymerase II is associated with nearly homogeneous yeast general initiation factor b by three criteria: cofractionation on the basis of size and charge and coinactivation by mild heat treatment. The kinase phosphorylates the CTD at multiple sites in a processive manner. Factor b may possess a DNA-dependent ATPase activity as well. Both kinase and DNA-dependent ATPase activities exhibit the same nucleotide requirements as previously demonstrated for the initiation of transcription. These results support the idea that phosphorylation of the CTD lies on the pathway of transcription initiation and identify a catalytic activity of a general factor essential for the initiation process.