Previous studies of bovine CYP11B1 gene regulation revealed six cis-acting elements, Ad1, Ad2, Ad3, Ad4, Ad5, and Ad6, in the 5' upstream region of the gene. Ad4 site was a positive transcription element in the stimulation by cAMP. Ad4-binding protein (Ad4BP) was purified from the nuclear extract of bovine adrenal cortex using affinity latex particles conjugated with polymerized Ad4 sequences. The molecular mass of the purified Ad4BP estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa. To characterize the binding specificity of Ad4BP, oligonucleotides homologous to Ad4 sequence and AGGTCA containing sequences in the promoter regions of steroidogenic P-450s were synthesized and used for gel shift analyses as competitors. The competition experiments revealed that Ad4BP bound not only to (C/T)CAAGG(T/C)(C/T), which was originally identified as the Ad4 binding site, but also to (Pu)PuPuAGGTCA. All the steroidogenic P-450 genes examined had at least one Ad4BP binding sequence. Experiments with model sequences containing various nucleotide substitutions established that (C/T)CAAGG(T/C)CA is the strongest binding sequence for Ad4BP. The expression of Ad4BP was examined with adrenal cortex cells and several other steroidogenic and nonsteroidogenic cells. Only the steroidogenic cells, the granulosa cells of bovine ovary, and I-10 cells derived from mouse Leydig cells, expressed the binding activity to Ad4 site. The presence of Ad4 site as a common cis-acting element in the genes of all the steroidogenic P-450s and the steroidogenic tissue-specific expression of Ad4BP strongly suggests that Ad4BP is an indispensable transcription factor for the expression of all the steroidogenic P-450 genes.