In our previous studies, we demonstrated that ERK1/2 (extracellular signal-regulated protein kinase) and p38 MAPK (mitogen-activated protein kinase) are required for gonadotropin-releasing hormone (GnRH)-II-induced anti-proliferation of ovarian cancer cells. In the present study, we examined the role of the GnRH-I receptor, as well as the activation of protein kinase C (PKC), in the anti-proliferative effect induced by GnRH-I or II in ovarian cancer cells. Our results demonstrated that Antide, a GnRH-I antagonist, reversed the activation of ERK1/2 induced by GnRH-I or II and abolished the anti-proliferative effect of GnRH-I and II in ovarian cancer cells. Transfection of short-interfering RNA to abrogate the gene expression of the GnRH-I receptor reversed GnRH-I and II-induced anti-proliferation. These results indicate that GnRH-I or II induce anti-proliferation through the GnRH-I receptor in ovarian cancer cells. In addition, the activation of ERK1/2 by GnRH-I or II was mimicked by phorbol-12-myristate 13-acetate, a PKC activator. Pretreatment with GF109203X, an inhibitor of PKC, blocked GnRH-induced ERK1/2 activation and anti-proliferation. These results suggest that the activation of PKC is responsible for GnRH-induced ERK1/2 activation and anti-proliferation in ovarian cancer cells. Taken together, these results indicate that binding of GnRH-I and II to the GnRH-I receptor activates ERK1/2 through a PKC-dependent pathway and is essential for GnRH-induced anti-proliferation of ovarian cancer cells.