In eukaryotes, initiation of mRNA synthesis is a multistep process that is carried out by RNA polymerase II and auxiliary factors that are commonly referred to as basal or general factors. In this study accurate initiation of transcription was reconstituted with purified, Escherichia coli-synthesized TFIIB, TBP (the TATA box-binding polypeptide of the TFIID complex), and the 30-kD subunit of TFIIF (also known as RAP30), along with purified, native RNA polymerase II from Drosophila embryos, calf thymus, or HeLa cells. This minimal set of factors was able to transcribe a subset of the promoters tested. The addition of both subunits of TFIIE and the 74-kD subunit of TFIIF increased the efficiency of transcription by a factor of 2 to 4. In contrast, the inclusion of a crude TFIID fraction from Drosophila embryos in place of recombinant TBP resulted in a strong dependence on TFIIE. By gel mobility-shift analysis, TFIIB, TBP, RAP30, and polymerase were able to assemble into DB and DBPolF30 complexes with transcriptionally competent (wild type or initiator mutant), but not with transcriptionally inactive (TATA and TATA/initiator mutant), versions of the Drosophila Adh promoter. Thus, it appears that RNA polymerase II is able to initiate transcription subsequent to assembly of the DBPolF30 complex, which is a minitranscription complex that represents the central core of the RNA polymerase II transcriptional machinery.