In the bovine ovary, thecal cells are the only cell type capable of expressing the CYP17 gene in response to LH. With the onset of ovulation and luteinization in the cow, there is complete loss of P450c17alpha expression. To characterize the molecular mechanisms involved in tissue-specific regulation of the CYP17 gene in the bovine ovary, deletion mutations of the bovine CYP17 promoter were ligated into a promoterless luciferase expression vector, and reporter constructs were transiently transfected into primary cultures of bovine thecal and luteal cells. Deletion of the promoter sequences between -191 and 101 bp dramatically decreased the levels of reporter gene activity in both thecal and luteal cells. Computer-assisted analysis revealed the presence of a putative inverted Sp1-like binding site at -188/-180 bp. Deletion or mutation of this sequence caused a decrease in both basal and forskolin-stimulated reporter gene activity. In addition, mutation or deletion of this sequence also decreased reporter gene expression induced by overexpression of the protein kinase A catalytic subunit. Electrophoretic mobility shift assays showed that this sequence binds to a nuclear protein(s) from both thecal and luteal cells that is related to Sp1, as suggested by the results of gel mobility supershift assay employing an antibody raised against Sp1. DNA-binding activity was not increased by the addition of forskolin to thecal or luteal cells. We conclude that this inverted Sp1-like binding sequence is involved in constitutive as well as cAMP-dependent expression of the CYP17 gene in the bovine ovary.