CRH-binding protein (CRH-BP) binds CRH with high affinity and inhibits CRH-mediated ACTH release from anterior pituitary corticotrope-like cells in vitro. In female mouse pituitary, CRH-BP is localized not only in corticotropes, but is also expressed in gonadotropes and lactotropes. To investigate the functional significance of gonadotrope CRH-BP, we examined the molecular mechanisms underlying GnRH-regulated CRH-BP expression in T3-1 gonadotrope-like cells. CRH-BP is endogenously expressed in T3-1 cells and quantitative real time RT-PCR and ribonuclease protection assays demonstrate that GnRH induces a 3.7 fold increase in CRH-BP mRNA levels. GnRH also induces intracellular CRH-BP (2.0 fold) and secreted CRH-BP (5.3 fold) levels, as measured by ¹²⁵I-CRH: CRH-BP chemical cross-linking. Transient transfection assays using CRH-BP promoter-luciferase constructs indicate that GnRH regulation involves PKC-, ERK- and calcium-dependent signaling pathways and is mediated via a multipartite GnRH response element that includes AP-1 and CRE sites. The CRE site significantly contributes to GnRH responsiveness, independent of PKA, representing a unique form of multipartite GnRH regulation in T3-1 cells. Furthermore, electrophoretic mobility shift assays indicate that T3-1 nuclear proteins specifically bind at AP-1 and CRE sites. These data demonstrate novel regulation of pituitary CRH-BP, highlighting the importance of the pituitary gonadotrope as a potential interface between the stress and reproductive axes.