Cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene expression is regulated by gonadotropins via cAMP in the ovary and by ACTH via cAMP in adrenal cortical cells. Previously, we have characterized a response element located at -118 to -101 bp in the 5'-flanking region of the bovine P450scc gene required for cAMP-stimulated transcription in both mouse adrenocortical Y1 cells and bovine ovarian cells in primary culture. It was shown that this region contains a binding site for the transcription factor Sp1. Deletion of this sequence abolished cAMP-stimulated transcription in both Y1 cells and bovine ovarian luteal cells. Another sequence element located at -57 to -32 bp upstream from the transcription initiation site, which is highly conserved in CYP11A of other species, contains the motif TAGCCTTG, similar to the consensus binding site of steroidogenic factor-1, SF-1 (or Ad4-BP), but in the inverted orientation. In the present study, gel shift analysis using nuclear extracts of either Y1 cells or bovine luteal cells demonstrated that the sequence between -57 and -32 bp bound SF-1. A mutation of the SF-1-binding site that abolished binding of the nuclear protein to DNA reduced markedly the basal transcription of the reporter gene as well as the responsiveness to cAMP, when the mutated fragments containing the region from -186 to +12 bp were cloned into a luciferase construct and transfected into mouse adrenal Y1 cells and bovine luteal cells. The role of SF-1 in P450scc transcription was further confirmed by transactivation of the -186/+12Luc construct employing an SF-1 expression vector after transfection into nonsteroidogenic COS-1 cells. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites, and from a construct containing both Sp1 and SF-1 elements upstream of the CYP11A TATA box, indicated that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y1 cells. Finally, a mammalian two-hybrid system was employed to demonstrate that Sp1 and SF-1 can associate in vivo. These results establish that basal and cAMP-stimulated activity of the bovine P450scc promoter in both Y1 cells and bovine luteal cells requires the combined action of at least two transcription factors, Sp1 and SF-1.