GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LT2 gonadotrope cells (maximum increase: 2.5 fold; EC₅₀: 0.30 nM) and this was further evidenced by GnRH activation of a cAMP sensitive reporter gene. GnRH effect was Ca⁺⁺ independent, mimicked by the phorbol ester PMA and blocked by the protein kinase C (PKC) inhibitor, bisindolylmaleimide, indicating that GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production whereas down-regulation of novel PKC, and by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKC or but not PKC impaired GnRH activation of a cAMP-sensitive promoter demonstrating that PKC and are the two endogenous isoforms mediating GnRH activation of adenylyl cyclase (AC) pathway in LT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP suggesting that in vivo, cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.