Although follicle-stimulating hormone (FSH) plays an essential role in controlling gametogenesis, the biology of FSH transcription remains poorly understood, but is known to involve the complex interplay of multiple endocrine factors including gonadotropin-releasing hormone (GnRH). We have identified a GnRH-responsive element within the rat FSH promoter containing an E-box and partial CRE site that are bound by the basic helix loop helix transcription factor family members, upstream stimulating factor (USF)-1/USF-2, and the basic leucine zipper member, cAMP response element binding protein (CREB), respectively. Expression studies with CREB, USF-1/USF-2, and activating protein-1 (AP-1) demonstrated that the USF transcription factors increased basal transcription, an effect not observed if the cognate binding site was mutated. Conversely, expression of a dominant negative CREB mutant or CREB knockdown attenuated induction by GnRH, whereas dominant negative Fos or USF had no effect on the GnRH response. GnRH stimulation specifically induced an increase in phosphorylated CREB (P-CREB) occupation of the FSH promoter, leading to the recruitment of CREB binding protein (CBP) to enhance gene transcription. In conclusion, a composite element bound by both USF and CREB serves to integrate signals for basal and GnRH-stimulated transcription of the rat FSH gene.