The asynchronous secretion of gonadotrope LH and FSH under the control of GnRH is crucial for ovarian cyclicity but the underlying mechanism is not fully resolved. Since prostaglandins (PG) are autocrine regulators in many tissues, we determined whether they have this role in gonadotropes. We first demonstrated that GnRH stimulates PG synthesis by induction of COX-2, via the PKC/c-Src/PI3K/MAPK pathway in the LT2 gonadotrope cell line. We then demonstrated that PGF₂ and PGI₂, but not PGE₂ inhibited GnRH receptor expression by inhibition of phosphoinositide turnover. PGF₂, but not PGI₂ or PGE₂, reduced GnRH-induction of LH gene expression, but not the GSU or the FSH subunit genes. The prostanoid receptors EP1, EP2, FP and IP were expressed in rat gonadotropes. Incubations of rat pituitaries with PGF₂, but not PGI₂ or PGE₂, inhibited GnRH-induced LH secretion, while the COX inhibitor, indomethacin, stimulated GnRH-induced LH secretion. None of these treatments had any effect on GnRH-induced FSH secretion. The findings have thus elaborated a novel GnRH signaling pathway mediated by PGF₂-FP and PGI₂-IP, which acts through an autocrine/paracrine modality to limit autoregulation of the GnRH receptor and differentially inhibit LH and FSH release. These findings provide a mechanism for asynchronous LH and FSH secretions and suggest the use of combination therapies of GnRH and prostanoid analogs to treat infertility, diseases with unbalanced LH and FSH secretion and in hormone-dependent diseases such as prostatic cancer.