We have analyzed the sequences that regulate the transcription of adenovirus type 5 early region 4 (E4). A region located immediately upstream of the E4 TATA box is required for efficient E4 transcription in vitro and in vivo. A cellular transcription factor, termed ETF-A, binds to this region. ETF-A also binds to additional sites in the E4 5'-flanking region, including the adenovirus terminal repeat, as well as to the adenovirus early region 2 promoter region and the adeno-associated virus early promoter region. A repeated sequence motif is found in each binding domain. The same factor binds to a region upstream of a cellular gene that contains a cyclic AMP response element. Consistent with this result, E4 expression is induced in vivo by cyclic AMP. Two other regions further upstream of the E4 initiation site also contribute to efficient E4 expression. These domains are functionally redundant and contain binding sites for ETF-A. One domain is the adenovirus terminal repeat, which has strong promoter activity in vitro and in vivo.