This study defines a DNA sequence upstream from the mRNA cap site required for in vivo expression of the adenovirus 2 pre-early region. The adenovirus 2 pre-early region and flanking sequences were cloned in Escherichia coli plasmid pBR322. Derivatives of the plasmid lacking portions of the upstream viral DNA sequence were constructed. An assay was devised to test the ability of these plasmid DNAs to complement an adenovirus 5 mutant with a deletion in the pre-early region. Plasmids that retained at least 38 base pairs upstream from the mRNA cap site had complementing activity similar to that of the original plasmid, which contains 229 base pairs of upstream viral sequence. However, plasmids retaining 23 or fewer base pairs of viral sequence upstream from the cap site had significantly reduced complementing activity. These results indicate that a portion of the adenovirus 2 sequence between 23 and 38 base pairs upstream from the mRNA cap site is required for expression of the pre-early region. This interval includes the Goldberg-Hogness box-like sequence T-A-T-T-T-A-T-A.