A proximal element from the human StAR gene promoter, containing the sequence (-105)TATCCTTGAC(-95), was shown to confer responsiveness to 8-Br-cAMP in the presence of steroidogenic factor 1 (SF-1) when placed behind a minimal thymidine kinase promoter or an SV40 promoter and transfected into BeWo cells which normally lack StAR and SF-1. This element was also transactivated by SF-1 in a yeast one-hybrid system. The -105 to -95 sequence was protected by SF-1 in footprint analysis and a double-stranded oligonucleotide containing the element bound SF-1 specifically in electrophoretic mobility shift assays. Another SF-1-binding sequence 35 bp upstream of the transcription start site ((-42)CAGCCTTC(-35)) was identified in the DNase 1 footprint analysis and, when mutated, markedly reduced SF-1-dependent and 8-Br-cAMP-stimulated StAR promoter activity in BeWo cells. The two proximal SF-1 response elements were shown to be critical for StAR promoter function in human granulosalutein cells, which express SF-1 and respond to cAMP with increased transcription of the StAR gene. Mutation of either element substantially reduced basal and forskolin-stimulated promoter activity, although mutation of the -105 to -95 element had more pronounced effects. Mutation of a third, more distal, SF-1-binding site at -926 to -918 also reduced basal but not forskolin-stimulated promoter activity in the granulosa-lutein cells. These findings demonstrate that multiple SF-1 response elements are required for maximal StAR promoter activity and regulation by cAMP.