Luteinizing hormone (LH) and insulin stimulate transcriptional activity of the porcine low density lipoprotein (LDL)-receptor promoter supraaddtively in primary cultures of granulosa-luteal cells. The mechanistic basis of this bihormonal interaction is unknown. Unlike the human, the pig LDL-R gene promoter has four imperfect repeats, which include two tandem upstream and one downstream putative Sp1/Sp3 binding sites and one canonical sterol-regulatory element (SRE) site between -255 to -139 bp 5'- upstream to the transcriptional start site. To assess the role of SRE binding protein (SREBP) in LDL-receptor gene regulation, swine granulosa-luteal cells were cotransfected with constitutively driven minigenes CMV/SREBP-1a or SREBP-2 and the pLDLR1076/luc promoter. SREBP-1a and SREBP-2 stimulated LDL-R gene transcription by 8- and 4-fold (both P < 0.01), respectively. LH alone augmented stimulation by SREBP-1 by 2-fold (P < 0.05). Conversely, cotransfection of a dominant-negative mutant form of SREBP-1a (lacking sequences for the N-terminal 90 amino acids) repressed basal and hormonally stimulated LDL-R promoter activity by > 80% (P < 0.01). The relevance of the SRE site in the LDL-receptor promoter was examined by mutation of the cognate -167 ATCACCCCATG -157 to -167 ATCACCgCATG -157 bp. This substitution decreased basal expression by 50% (P < 0.05) and LH and insulin/IGF-I-stimulated transcriptional activity by 80% and > 90% respectively (both P < 0.01). Mutations within each of the three flanking putative Sp1/Sp3 sites at - 216 TCCTCC -211, - 201 TCCTCC - 196 and - 151 TCCTCC -146 bp in the LDL-receptor gene promoter also reduced basal activity (by > 85%, P < 0.05) and hormonal responsiveness (by > 95%, P < 0.05). Electrophoretic gel mobility-shift assays (EMSA) using swine granulosa-luteal cell nuclear protein with specific antibody or recombinant SREBP-1a protein confirmed that presumptive SRE-1 and Sp1/Sp3 elements bind respective peptides. Motifs that putatively associate with Sp1/Sp3 appear to be important functionally, since mithramycin, an inhibitor of Sp1/Sp3 protein(s) binding, blocked hormonally induced LDL-receptor promoter expression by 80% (P <0.01). In conclusion, basal transcription and supraadditive stimulation of porcine LDL-receptor gene transcription by LH and insulin in granulosa-luteal cells requires SREBP-1a and Sp1/Sp3 binding elements.