Estrogen, acting via ER, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ER signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ER DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and independent estrogen signaling in estrogen regulation of LH, FSH, prolactin and Activin/Inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LH mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary GSU, FSH and ActivinB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ER signaling suppresses serum LH via reduced secretion not synthesis. Additionally, ERE-dependent and ERE-independent ER pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.