Cytochrome P450scc (CYP11A1) is the enzyme that catalyzes the side-chain cleavage reaction of cholesterol, the first and rate-limiting reaction in the biosynthesis of steroid hormones in the adrenal cortex. DNase-I-footprinting analysis using nuclear extracts from the bovine adrenal cortex and the 5′ upstream regulatory region (nucleotides –1697 to – 1523) of the CYP11A1 gene, which is mainly required for response to cAMP [Inoue, H., Watanabe, N., Higashi, Y. & Fujii-Kuriyama, Y. (1991) Eur. J. Biochem. 195, 563–569], revealed that some protein factors bound to that region. One of the sequences protected by the binding factors is a cAMP-responsive-element (CRE)-like sequence, which is known to be recognized by CRE-binding protein (CREB) or its related proteins, and another is a sequence designated Ad4 which is bound by a tissue-specific factor, Ad4-binding protein (Ad4BP). The region containing the two closely arranged DNA sequences showed a high level of cAMP responsive and cell-specific expression when it was fused to the basal promoters. Introduction of point mutations in these sequences demonstrated that the CREB/ATF factors and Ad4BP bound to the sequences showed synergistic enhancer effects on cAMP-responsive and cell-specific expression of the CYP11A1 gene.