We have studied promoter opening in assays reconstituted with purified RNA polymerase II and basal transcription factors. We found that creating a region of heteroduplex DNA around the start site of the adenovirus major late (AdML) promoter circumvents the requirement for TFIIE and TFIIH in transcription. The critical size and position of the heteroduplex region that alleviates the requirement for TFIIE and TFIIH is six nucleotides, from -4 to +2. Promoter opening was investigated directly with potassium permanganate (KMnO4), a chemical probe specific for single-stranded thymidines. We found that KMnO4-detectable opening of the AdML promoter requires the presence of the complete pre-initiation complex, DBpolFEH, and that opening occurs in two discrete steps. First, dependent on ATP but prior to initiation, the -9 to +1 region becomes single-stranded. Second, formation of the first phosphodiester bond results in expansion of the open region to the +8 position. Our results lead to a model in which the critical function of the TFIIH-associated DNA helicases is to create a single-stranded region. This gives RNA polymerase II access to the nucleotides of the template strand and allows expansion of the open region upon formation of the first phosphodiester bond.