An in vitro system [Weil, P. A., Luse, D. S., Segall, J. & Roeder, R. G. (1979) Cell 18, 469-484 and Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L., (1980) Proc. Natl. Acad. Sci USA 77, 3855-3859] was adapted for studying initiation of transcription of the ovalbumin gene. The DNA template was a cloned ovalbumin gene fragment that contained 5' flanking sequences, the first structural sequence region, and a portion of the first intervening sequence. A HeLa cell crude extract was used as the source of RNA polymerase and initiation factors. Correct initiation was judged by the sizes of the transcription products generated from ovalbumin templates truncated at various positions before the 3' end of the gene. Transcription of the specific product was carried out by RNA polymerase II, as judged from alpha-amanitin sensitivity. A series of deletion mutants was constructed by trimming 5' flanking sequences of the ovalbumin DNA template by using exonuclease III. The DNAs generated were then cloned in pBR322 and used as templates to determine which sequences were necessary for initiation of transcription. Specific initiation of the ovalbumin gene was unaffected by deletion of all but 61 nucleotides of the 5' flanking sequence but completely abolished by deletion of all but 26 nucleotides of 5' flanking sequence. Thus, a region between 61 and 26 nucleotides upstream from the cap site, which includes the Hogness box (T-A-T-A-T-A-T) at position 32-26, is essential for the correct initiation of the ovalbumin gene. Nevertheless, natural DNA fragments containing false Hogness boxes (T-A-T-A-A-A-A and T-A-T-A-T-A-T) not normally located in the immediate 5' flanking region of an authentic gene did not serve as promoters for initiation of transcription. These results suggest that the Hogness box is essential, but not sufficient, for specific initiation of RNA synthesis.