THE general transcripton factor TFIIE, together with other general transcription factors, is essential for transcription initiation by RNA polymerase II1-5 TFIIE stimulates the TFIIH-dependent kinase activity that phosphorylates the carboxy-terminal domain of the largest subunit of RNA polymerase II6, and possesses a helicase activity(7). Here we show that human TFIIH has DNA-dependent ATPase activity and we characterize the stimulatory effect of TFIIE on both the ATPase and kinase activities. We demonstrate that extensive phosphorylation of RNA polymerase II occurs in a TFIIE-dependent manner in both the absence and presence of DNA but, in the latter case, only at a late stage of preinitiation complex assembly. We also show that TFIIH specifically phosphorylates three general transcription factors, human TFIID tau (TBP), TFIIE-alpha and TFIIF-alpha (RAP74).