An RNA polymerase transcription factor IIH holoenzyme (holoTFIIH) has been resolved to near homogeneity from Saccharomyces cerevisiae. HoloTFIIH comprises the five-subunit core transcription factor described previously (Feaver, W. J., Svejstrup, J. Q., Bardwell, A. J., Bardwell, L., Buratowski, S., Gulyas, K. D., Donahue, T. F., Friedberg, E. C. and Kornberg, R. D. (1993) Cell 75, 1379-1387) and in addition, SSL2 and three further, as yet unidentified, polypeptides. HoloTFIIH possesses C-terminal repeat domain kinase activity and, together with other pure yeast transcription proteins, enables RNA polymerase II transcription in a fully defined system. By contrast, core TFIIH is inert in both C-terminal repeat domain kinase and reconstituted transcription assays.