Gonadotropin-releasing-hormone (GnRH) is a key regulator of the reproductive system. Binding of GnRH to its receptor (GnRHR), a member of the G-protein-coupled-receptor (GPCR) family, leads to activation of the PLCβ-Ca2+-PKCs-MAPKs cascade, culminating in gonadotropins (LH and FSH) synthesis and release. The role of PI3K and PI4K in GnRH-induced ERK1/2 activation was investigated in αT3-1 and LβT2 gonadotrope cells. αT3-1 and LβT2 cells were preincubated for 1h with Wortmannin (10nM and 10µM), or LY294002 (10µM and 100µM), doses known to inhibit PI3K and PI4K, respectively, followed by stimulation with GnRH (100nM) or PMA (100nM) for 5 or 10min. We found a significant inhibition of ERK1/2 activation by GnRH or PMA at the two doses of Wortmannin examined, with a more pronounced inhibition observed in the more mature LβT2 cells. We also found a significant inhibition of ERK1/2 activation by GnRH or PMA at the two doses of LY294002 examined, with a more pronounced inhibition observed in the αT3-1 cells. On the other hand, we found no inhibition of ERK1/2 activation by GnRH or PMA when cells were preincubated with Wortmannin or LY294002 for 30min. Furthermore, Wortmannin (10nM and 10µM) inhibited also GnRH- induced MEK phosphorylation (Ser298), an upstream effector of ERK1/2, in LβT2 cells. GnRH-induced Akt activity, which is a downstream target of PI3K, was also examined. When αT3-1 cells were treated with GnRH (100nM), we noticed that the basal phosphorylation of Akt in both sites (Ser473, Thr308) was markedly high and was rapidly reduced by GnRH within 5min of stimulation. We conclude that the lack of inhibition of GnRH-induced ERK1/2 activation by Wortmannin and LY294002 at 30min raises the interesting possibility that the role of PI3K/PI4K is indirect and exerted by a factor which needs to be identified. Furthermore, the involvement of PI3K/PI4K in ERK1/2 activation by GnRH is independent of Akt activity.