The GATA family of transcription factors are critical determinants of cell differentiation as well as regulation of adult gene expression throughout the reproductive axis. Within the anterior pituitary gland, GATA factors have been shown to increase glycoprotein a-subunit gene promoter activity; however, nothing has been known about the impact of these factors on expression of the gonadotropin b-subunits. In this study, we demonstrate expression of both GATA2 and GATA4 in primary mouse gonadotropes and the gonadotrope cell line, LbT2. Based on the transient transfection in fibroblast cells, GATA factors increase LH b-subunit gene (LHb) promoter activity alone and in synergy with the orphan nuclear receptors steroidogenic factor-1 (SF-1) and liver receptor homologue-1 (LRH-1). The GATA response was localized to a DNA regulatory region at position K101 in the rat LHb gene promoter which overlaps with a previously described cis-element for pituitary homeobox-1 (Pitx1) and is flanked by two SF-1/LRH-1 regulatory sites. As determined by gel shift, GATA and Pitx1 can compete for binding to this element. Furthermore, mutation analysis revealed a requirement for both the GATA/Pitx1 and the SF-1/LRH-1 cis-elements in order to achieve synergy. These studies identify a novel role for GATA transcription factors in the pituitary and reveal additional molecular mechanisms by which precise modulation of LHb gene expression can be achieved.