The FSH receptor (FSHR) is selectively expressed in the granulosa and Sertoli cells in a development-dependent manner. Little is known regarding how the regulatory factors balance expression of this gene in ovarian cycles or spermatogenic stages. We have used the ovine FSHR promoter as a model system and identified a third regulatory element (RE-3) located at −197 to −171 of the strongest promoter. Gel mobility shift and antibody supershift assays demonstrated that nuclear factors c-Fos/c-Jun, steroidogenic factor-1 (SF-1), upstream stimulatory factor-1/2 (USF-1/2), and chicken ovalbumin upstream promoter transcription factor-1/2 (COUP-TFI/II) potentially bound to RE-3. We have also extended our previous observations by showing that a sequence containing an E-box was not only bound by USF proteins but also recognized by COUP-TF orphan receptors. Functional studies demonstrated that USF-1/2, c-Fos/c-Jun, and SF-1 were activators, whereas COUP-TFs were repressors. Our studies indicated that RE-3 mediated SF-1 activation as well as phorbol 12-myristate 13-acetate stimulation, whereas COUP-TFs inhibited AP-1, USFs, and SF-1 activation. We also demonstrated that both COUP-TF-binding sites in the core promoter were required for the bipartite elements to oppose their competitor binding. These data suggest a mechanism by which positive and negative regulators compete for the common regulatory elements, providing antagonistic pathways that might govern the expression of FSHR in gonadal cells.