Efficient replication of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) requires the virus transactivator proteins known as Tat. In order to understand the molecular mechanisms involved in Tat transactivation, it is essential to identify the cellular target(s) of the Tat activation domain. Using an in vitro kinase assay, we previously identified a cellular protein kinase activity, Tat-associated kinase (TAK), that specifically binds to the activation domains of Tat proteins. Here it is demonstrated that TAK fulfills the genetic criteria established for a Tat cofactor. TAK binds in vitro to the activation domains of the Tat proteins of HIV-1 and HIV-2 and the distantly related lentivirus equine infectious anemia virus but not to mutant Tat proteins that contain nonfunctional activation domains. In addition, it is shown that TAK is sensitive to dichloro-1-beta-D-ribofuranosylbenzimidazole, a nucleoside analog that inhibits a limited number of kinases and is known to inhibit Tat transactivation in vivo and in vitro. We have further identified an in vitro substrate of TAK, the carboxyl-terminal domain of the large subunit of RNA polymerase II. Phosphorylation of the carboxyl-terminal domain has been proposed to trigger the transition from initiation to active elongation and also to influence later stages during elongation. Taken together, these results imply that TAK is a very promising candidate for a cellular factor that mediates Tat transactivation.