Expression of the FSH receptor (Fshr) is restricted to testicular Sertoli cells and ovarian granulosa cells, thereby limiting the direct targets of FSH action to these somatic cells of the gonads. Earlier studies indicate that transcription of Fshr in the gonads requires elements outside the gene's immediate 5' flanking sequence. To help uncover candidate regulatory sequences comparative genomics and DNase I hypersensitivity mapping were employed. 156 evolutionarily conserved sequences were found and partial DNase I hypersensitivity mapping across 45kb of 5' flanking sequence and the first intron identified four hypersensitive sites, DHS#1-#4. Notably, DHS#1and DHS#2 localized to conserved sites in the promoter region and exon 1 and correlated with the active state of the gene. DHS#3 also corresponded to a conserved site (site 7) but was more pronounced in non-expressing myoid cells, suggesting a role in gene silencing. Transient transfection analysis of DHS#3 confirmed its role in gene silencing, a function that was promoter, cell-type and position dependent. Protein-DNA binding studies on DHS#3 revealed that OCT-1 and GATA-4 bound site 7, in vitro, and transient transfection analysis showed that their binding sites were required for silencing activity. Furthermore, chromatin immunoprecipitation (ChIP) revealed that OCT-1 bound to site7 in the endogenous gene, but only in myoid cells. In contrast, GATA-1 bound site7 predominantly in Sertoli cells, suggesting it attenuates silencer activity. The findings reveal that OCT-1 binds within DHS#3 to silence Fshr transcription and implicate members of the GATA family in the modulation of this activity.