Gonadotropin-releasing hormone (GnRH) acts on its cognate receptor in pituitary gonadotropes to regulate the biosynthesis and secretion of gonadotropins. This action is thought to be mediated by the receptor coupling to G_q/11. GnRH also has extra-pituitary functions including inhibition of cell growth in prostatic, breast and ovarian cancers, in which the GnRH receptor activation of the MAP kinase cascades plays an important role. In extra-pituitary compartments, GnRH receptor signaling is potentially mediated by different G proteins such as G_i/o or G_s, although this remains controversial. Here we examined the GnRH receptor coupling to different G proteins and their roles in the activation of the MAP kinases. In MCF-7 and HEK293 cells transiently transfected with GnRH receptor cDNA, pertussis toxin had little effect on ERK activation. In contrast, ERK activation was completely abolished by a Gα_q selective inhibitor. In HEK293 cells co-transfected with GnRH receptor cDNA and siRNA against Gα_q/11, GnRH-stimulated ERK activation was markedly reduced. In Gα_q/11-negative mouse embryonic fibroblasts (MEF) stably expressing the rat GnRH receptor, no GnRH-stimulated ERK or JNK activation was observed. After transient transfection with Gα_q cDNA, GnRH stimulated phosphorylation of ERK and JNK. Using specific inhibitors, ERK activation was shown to occur through a classical Gα_q/11-PLC_β-PKC dependent system with consequent activation of c-Src, Raf-1 Kinase and MEK 1/2. Transient transfection of all subtypes of Gα_i or Gα_qi5 into the MEF stable cells did not facilitate GnRH-stimulated ERK activation. In addition, no GnRH-mediated cAMP response or inhibition of isoproteranol-induced cAMP accumulation in COS-7 and MEF cells was observed. Interestingly, GnRH-stimulated cleavage of poly[ADP-ribose]polymerase (PARP), a substrate of caspase 3, was detected after transfection of Gα_q. We therefore propose that the mammalian GnRH receptor does not couple directly to Gα_i or Gα_s as hypothesised by other studies, that ERK and JNK activation by the GnRH receptor is mainly mediated via the Gα_q signalling pathway and that this pathway may play important roles in GnRH analogue-induced antiproliferation and apoptosis in the cell lines tested.