In this study we examined agonist-induced internalization of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by receptor binding assay on viable cells and confocal microscopy. The agonists nociceptin/orphanin FQ (NC), NC-NH(2), NC(1-13)-NH(2), [(pF)Phe(4)]NC-NH(2) and RO 64-6198 promote a rapid, concentration-dependent internalization of the hNOP receptor. Under the same conditions, [Phe(1),psi(CH(2)NH)Gly(2)]NC(1-13)-NH(2) and [Phe(1), psi(CH(2)NH)Gly(2),Arg(14),Lys(15)]NC(1-13)-NH(2) failed to induce significant, concentration-dependent NOP receptor endocytosis; even when present at high concentrations (up to 1 mM) they promoted only an approximately 25-30% internalization of hNOP receptors. We also investigated hNOP receptor desensitization upon agonist challenge: ligand efficacy to inhibit forskolin-stimulated cAMP production. After 1 h exposure to NC, NC-NH(2), NC(1-13)-NH(2), [(pF)Phe(4)]NC-NH(2) and RO 64-6198 (5 microM) = 20 to 30% of receptor desensitization was observed. Moreover, we found that the blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. The non-internalizing agonists [Phe(1),psi(CH(2)NH)Gly(2)]NC(1-13)-NH(2) and [Phe(1), psi(CH(2)NH)Gly(2),Arg(14),Lys(15)]NC(1-13)-NH(2) (100 microM) resulted in a strong (67 and 74 %, respectively) receptor desensitization which was not influenced by monensin. Finally, CHO-hNOP cells exposed to the receptor-internalizing agonists for 24 h resulted in a significantly higher cAMP accumulation (defined supersensitization) compared with the non-internalizing agonists. In addition, blocking of receptor recycling by monensin led to a decrease of the cAMP accumulation only in cells exposed to internalizing agonists. These data show that prolonged receptor signaling mediated by receptor endocytosis and recycling/reactivation might reduce the development of tolerance but can enhance compensatory mechanisms that lead to supersensitivity of specific signaling pathways.