The adrenocorticotropin receptor or ACTH-R which is the type 2 among the melanocortin receptor family is almost exclusively expressed in the adrenal cortex, reflecting a high degree of tissue specificity. In human cultured adrenocortical cells, we have previously reported that ACTH in contrast to most of the peptide hormones, is able to up-regulate the number of its own receptors through an increase of the transcriptional activity of the encoding gene. Three putative SF-1 binding sites are present in the sequence of the human ACTH-R gene promoter at -35 (SF-35), -98 (SF-98) and -209 (SF-209). By EMSA studies, we demonstrated that these sites effectively bind SF-1 protein. After transient transfection of H295R cells using a construct containing the first 263 bp upstream of the transcriptional start site, in front of the luciferase gene in the pGL3 vector, we demonstrated the involvement of all three SF-1 sites to confer maximal constitutive activity to a proximal region of the hACTH-R gene promoter. Only SF-35 and SF-98 play a role in cAMP-induced regulation of this gene.