We present a physical and molecular genetic characterization of Drosophila melanogaster TFIIE (dTFIIE), a component of the basal RNA polymerase II transcription apparatus. We have purified dTFIIE to near homogeneity from nuclear extracts of Drosophila embryos and found that it is composed of two subunits with apparent molecular weights of 55 and 38 kDa. Peptide sequence information derived from the two subunits was used to isolate the corresponding cDNA clones, revealing that dTFIIE and human TFIIE share extensive amino acid similarity. Functional conservation was demonstrated by the ability of bacterially expressed dTFIIE to substitute for human TFIIE in an in vitro transcription assay reconstituted from purified components. Cytological mapping analysis shows that both subunits are encoded by single copy genes located on chromosome III.