Steroidogenic acute regulatory protein (StAR) is required for efficient adrenal cortical and gonadal but not trophoblast steroid hormone synthesis. StAR gene expression in gonadal cells is stimulated by tropic hormones acting through the intermediacy of cAMP. DNA sequence analysis of the human StAR gene promoter revealed two motifs resembling binding sites for steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor transcription factor family that controls expression of steroidogenic hydroxylases. The 5'-most sequence (distal site) is a consensus SF-1 binding site. The proximal site is a consensus estrogen receptor binding half-site. The StAR gene promoter is not active in BeWo choriocarcinoma cells, COS-1 cells, HeLa cells, or SK-OV-3 ovarian adenocarcinoma cells, all of which do not express significant levels of SF-1 mRNA. Introduction of SF-1 into these cells stimulated StAR promoter activity, particularly in response to cAMP. Two orphan nuclear transcription factors that bind to sequences similar to SF-1 sites, NGFI-B/Nur77 and RNR-1, did not support cAMP-stimulated StAR promoter activity in BeWo cells. Mutation of the distal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity in BeWo cells by 82% and 71%, respectively. Mutation of the proximal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity by 89% and 96%, respectively. Mutations in both sites reduced basal promoter activity to 7% of wild type promoter activity and cAMP-stimulated promoter activity to less than 5% of the wild type. Deletion analyses of promoter activity were consistent with the mutation studies. Electrophoretic mobility shift assays (EMSAs) demonstrated that the distal site binds to SF-1 expressed in COS-1 cells and to an SF-1-GST fusion protein with high affinity, but that the mutated distal sequence does not. An anti-SF-1 antibody ablated the characteristic SF-1-DNA complex with the distal sequence. The proximal site formed a number of protein-DNA complexes with COS-1 cell extracts, but appeared to have at best only very modest affinity for SF-1. Collectively, our findings demonstrate that SF-1 plays a key role in controlling the basal and cAMP-stimulated expression of the StAR gene. SF-1 can function at two distinct sites in the human StAR gene promoter, apparently by two different types of interaction, to control transcription.