Adrenocorticotropic hormone receptor (ACTHR) is expressed predominantly in the adrenal glands, and its expression is upregulated by its own ligand, ACTH, via a cAMP-dependent pathway. In the present study, we characterized the 5'-regulatory region of human ACTHR gene to elucidate the molecular mechanisms underlying its adrenal-specific and ACTH/cAMP-dependent expression. The promoter region (-1017/+47 when the transcription start site is regarded as +1) and its serial 5'-deletions (-764/+47, -503/+47, -214/+47 and -561+47) were ligated into the upstream of a luciferase (luc) reporter gene. These constructs were transfected into adrenocortical Y1 cells or non-adrenal JEG3 and Cos-1 cells. In all the cell lines, the luc activity gradually increased with serial 5'-deletions and the maximum activity was conferred by -56/+47. However, the magnitude of luc activity of each deletion construct in non-adrenal cells was much less than that in Y1 cells, suggesting that the promoter functions in an adrenal-specific manner. We identified two Steroidogenic Factor (SF)-1-binding sites at -209 and -35. Electrophoretic mobility shift assay (EMSA) demonstrated that both sites bind to SF-1. Mutation of both sites significantly decreased the activity of -214/+47 promoter in Y1 cells. Transfection of SF-1-expressing plasmid into non-adrenal cells significantly increased the promoter activity, suggesting that SF-1 plays a role in the tissue-specific expression of human ACTHR gene. We identified the region, -764 to -503, that was required for the for- skolin/camp responsiveness of the promoter. This region contains one AP-1 site. EMSA revealed that the binding of AP-1 to this site increased significantly upon treatment of Y1 cells with forskolin. Mutation of the site abolished the forskolin-responsiveness. In non-adrenal cells, the forskolin-responsiveness was observed only when SF-1-expressing plasmid was cotransfected. This is the first demonstration that both AP-1 and SF-1 are required for the cAMP-dependent induction of human ACTHR gene.