Regulated synthesis of luteinizing hormone (LH) requires coordinated transcriptional control of the a and LHβ subunits in pituitary gonadotropes. Several cis-acting elements and trans-acting factors have been defined for control of the LHβ promoter through heterologous cell culture models. In this report, we describe the identification of bipartite NF-Y (CBF/CP1) binding sites within the proximal bovine LHβ promoter. When multimerized, one of these sites activates the heterologous, minimal HSV thymidine kinase promoter in the gonadotrope-derived cell line αT3-1. The functional role of the promoter-distal site in regulating the full-length bovine LHβ promoter was assessed in vivo using transgenic mice harboring a mutant promoter linked to the chloramphenicol acetyltransferase reporter gene. While this element is important for conferring high level activity of the LHβ promoter in pituitary, it does not appear to be essential for mediating gonadotropin- releasing hormone (GnRH) regulation. This is the first characterization of a cis-acting element within this GnRH-dependent promoter that is restricted to regulating basal expression and not GnRH-induced activity.