Yeast RNA polymerase II initiation factor e was purified to homogeneity and identified by biochemical criteria as the counterpart of human transcription factor IIB. Factor e was essential for initiation of transcription from yeast and mammalian promoters in a reconstituted yeast transcription system. Activity resided in a single polypeptide of approximately 41 kDa, identified by peptide sequence analysis as the product of the SUA7 gene. Factor e interacted specifically with RNA polymerase II, consistent with a proposed role in determining the start site of transcription.