The activity of the N-terminal activation function AF-1 of RARα1 is abrogated upon mutation of a phosphorylatable serine residue (Ser-77). Recombinant RARα was phosphorylated by a variety of proline-directed protein kinases in vitro. However, only the coexpression of cdk7 stimulated Ser-77 phosphorylation in vivo and enhanced transactivation by RARα, but not by a S77A RAR mutant. Both free CAK (cdk7, cyclin H, MAT1) and the CAK-containing general transcription factor TFIIH phosphorylated Ser-77 in vitro. Furthermore RARα bound free CAK and purified TFIIH in vitro, and RARα–TFIIH complexes could be isolated from HeLa nuclear extracts. These findings represent the first example of activation of a transactivator through binding to and phosphorylation by a general transcription factor.