Transcription from the major late promoter of adenovirus type 2 DNA (including DNA sequences from 56 nucleotides upstream to 33 nucleotides downstream of the CAP site) was reconstituted with transcription factors purified from HeLa cells. Five components, transcription factors (TF) IIA, -B, -E, -D and RNA polymerase II, were required for accurate initiation of transcription. Kinetic analyses combined with order of addition experiments suggested that TFIIA acted first during the initiation reaction and that this interaction was followed by the action of TFIID. In agreement with these conclusions, both TFIIA and TFIID were required to render a transcription reaction partially resistant to concentrations of Sarkosyl previously shown to inhibit an early step in the formation of a preinitiation complex. Related Sarkosyl studies indicated that the inferred complex was subsequently recognized by RNA polymerase II, which resulted in an increased level of Sarkosyl-resistant transcription (in the presence of TFIIA and TFIID), and that this interaction occurred independently of TFIIB and TFIIE. However, TFIIB and TFIIE were implicated, along with the other factors and RNA polymerase II, in the subsequent formation of a highly stable preinitiation complex, which was inferred from its ability to initiate (with added nucleotides) in the presence of heparin concentrations which blocked unbound factors. The identification of a new transcription factor, which was required only when viral sequences 3' to the major late promoter were part of the transcription unit, is also reported.