The mouse gene encoding adrenal steroid 11 beta-hydroxylase (11 beta-OHase) has been cloned and the nucleotide sequence of its 5' end has been determined. The coding regions sequenced are homologous (75%) to the sequence of bovine 11 beta-OHase cDNA. The 5'-flanking region of the 11 beta-OHase gene contains a potential cAMP response element (TGACGTGA) located 56 base pairs upstream of the transcription initiation site (position -56) and two motifs at positions -249 and -148 which are similar to an element postulated to be required for the expression of 21-hydroxylase. Transfection of mouse Y1 adrenocortical tumor cells and MA-10 testicular Leydig cells with plasmids containing the 11 beta-OHase promoter linked to a growth hormone reporter gene showed that the 11 beta-OHase promoter can direct cell-specific expression. Deletion analyses of the 5'-flanking region suggest that multiple sequence elements, one of which is located between positions -425 and -338 and a second between positions -338 and -123, interact to produce full levels of promoter activity. Mutant Y1 cells defective in cAMP-dependent protein kinase activity do not express growth hormone driven by the 11 beta-OHase promoter, indicating that expression of 11 beta-OHase in Y1 cells requires an intact cAMP second messenger system. Moreover, mutation of the putative cAMP response element at position -56 abolishes expression. These experiments thus present a useful system for the investigation of cis-acting elements involved in the transcriptional regulation of 11 beta-OHase.