The effect of nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for the newly identified opioid receptor-like (ORL1) receptor, on mitogen-activated protein kinase (MAPK) was investigated in Chinese hamster ovary cells stably expressing ORL1 receptor. N/OFQ rapidly stimulated phosphorylation and activity of MAPK (p42 and p44 isoforms) in a concentration-dependent manner. The p42 isoform was preferentially activated by N/OFQ. Maximal activation (5.4 ± 1.2-fold of basal for p42 isoform) was achieved after a 1-min exposure of cells to 100 nM N/OFQ. The activation was blocked completely by pretreatment with pertussis toxin, but was not reversed by naloxone. U-73122, a phospholipase C-specific inhibitor, significantly inhibited phospholipase C activity, as well as MAPK activation stimulated by N/OFQ. Furthermore, N/OFQ-stimulated MAPK activation was suppressed by a protein kinase C-specific inhibitor, chelerythrine. The results demonstrate that N/OFQ can effectively stimulated MAPK by the activation of ORL1 receptor and pertussis toxin-sensitive G proteins, and that phospholipase C, as well as protein kinase C, is critically involved in these processes.