Estrogen receptors (ERs) are activated by their ligands as well as by signaling pathways that alter ER phophorylation in response to peptide hormones and growth factors. In pituitary gonadotrophs, gonadotropin releasing hormones (GnRHs) act via the type I GnRH receptor (GnRHR). Both GnRH subtypes (GnRH-I and GnRH-II) activate an estrogen response element (ERE)-driven luciferase reporter gene in LT2 mouse pituitary cells, and GnRH-I is most potent in this regard. Moreover, antide (a GnRH antagonist) and a GnRHR siRNA abrogate this effect, while an ER antagonist (ICI 182,780) does not. The ER in LT2 cells is phosphorylated at Ser¹¹⁸ in the nucleus and at Ser¹⁶⁷ in both nucleus and cytoplasm after GnRH treatments, coincided with increased ER binding to its co-activator, the p300/CBP-associated factor (PCAF). Moreover, siRNA-mediated knockdown of PCAF levels attanuated GnRH-induced ERE-luciferase trans-activation in these cells. Most importantly, both GnRH subtypes robustly up-regulated expression of the immediate early response gene, fosB, while co-treatment with ER siRNA or PCAF siRNA attenuated this effect. This appears to occur at the transcriptional level because co-recruitment of ER and PCAF to an ERE within the endogenous fosB promoter was increased by GnRH treatments, as shown by chromatin immunoprecipitation assays. These data demonstrate that GnRH-mediated phosphorylation of ER in mouse LT2 pituitary cells results in its rapid association with PCAF and the transcriptional activation of fosB, and we demonstrate that this in turn likely activates other genes in pituitary cells including the follicle-stimulating hormone subunit gene.