We have previously shown that early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) β deficiency. Egr-1 activates the LHβ gene in vitro through synergy with steroidogenic factor-1 (SF-1), a protein required for gonadotrope function. To test if this synergy is essential for gonadotropin-releasing hormone (GnRH) stimulation of LHβ, we examined the activity of the LHβ promoter in the gonadotrope cell line LβT2. GnRH markedly stimulated the LHβ promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHβ promoter attenuated this stimulation, whereas mutation of both promoter elements abrogated GnRH induction of the LHβ promoter. Furthermore, GnRH stimulated Egr-1 but not SF-1 expression in LβT2 cells. Importantly, overexpression of Egr-1 alone was sufficient to enhance LHβ expression. Although other Egr proteins are expressed in LβT2 cells and are capable of interacting with SF- 1, GnRH stimulation of Egr-1 was the most robust. We also found that the nuclear receptor DAX-1, a repressor of SF-1 activity, reduced Egr-1-SF-1 synergy and diminished GnRH stimulation of the LHβ promoter. We conclude that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHβ gene and plays a central role in the dynamic regulation of LHβ expression.