Fractionation of yeast whole-cell extract has led to the identification and purification of four novel proteins required for promoter-dependent transcription by RNA polymerase II in vitro. These initiation factors have been shown to be related in structure and function to RNA polymerase II initiation factors purified from mammalian cells. When combined with the well known TATA-binding protein, the entire set of initiation factors proves necessary and sufficient for accurate initiation from a variety of eukaryotic promoters. Studies with this defined transcription system have revealed the existence of additional proteins required for transcriptional activation or derepression by enhancer-binding regulatory proteins.