To further elucidate the mechanism of transcriptional initiation, we used synthetic oligonucleotides to prepare templates containing heteroduplex regions of varying size and location along the DNA of the adenovirus major late promoter. Unlike closed, linear DNA, or DNA with a downstream mismatch, DNA with a mismatch upstream of the initiation site only required the general factors TATA box-binding protein and transcription factor (TF) IIB to direct specific and accurate initiation in vitro by calf thymus RNA polymerase II. In the presence of TFIIF, initiation was possible on closed, linear DNA, but an upstream mismatch region still stimulated transcriptional initiation by more than 100-fold, leading to production of approximately 0.5 transcript/template in the absence of TFIIE, TFIIH, or ATP. The presence of a DNA mismatch was most effective in the -9 to -1 region; furthermore, stimulation by a mismatch did not require that the initiation site be included in the heteroduplex region. Efficient initiation at the immunoglobulin heavy chain promoter in the presence of TATA box-binding protein and TFIIB was also achieved when a mismatch region was introduced from -9 to +3. Our results suggest that initiation by RNA polymerase II in the absence of transcriptional activation is limited by melting of the promoter DNA upstream of the initiation site.