AbstractA synaptosomal preparation was employed to pharmacologically characterize the role of presynaptic nociceptin/orphanin FQ (N/OFQ) receptors (NOP receptors) in the regulation of 5‐hydroxytryptamine release in the Swiss mouse neocortex. In the present study, the NOP receptor ligands N/OFQ, Ac‐RYYRWK‐NH2 and [Phe1ψ(CH2‐NH)Gly2]N/OFQ(1–13)‐NH2 inhibited the K+‐induced [3H]‐5‐HT overflow with similar maximal effects (≈−35%) but different potencies (pEC50 of 8.56, 8.35 and 7.23, respectively). The novel agonist [Arg14,Lys15]N/OFQ also inhibited [3H]‐5‐HT overflow, but the concentration–response curve was biphasic and the efficacy higher (≈−45%). Receptor selectivity of NOP receptor agonists was demonstrated by showing that synaptosomes from NOP receptor knockout mice were unresponsive to N/OFQ, [Arg14,Lys15]N/OFQ and [Phe1ψ(CH2‐NH)Gly2]N/OFQ(1–13)‐NH2 but maintained full responsiveness to endomorphin‐1. Moreover, the inhibitory effect of N/OFQ was prevented by peptide ([Nphe1]N/OFQ(1–13)‐NH2 and UFP‐101) and nonpeptide (J‐113397 and JTC‐801) NOP receptor selective antagonists. Desensitization occurred under perfusion with high (3 and 10 µm) N/OFQ concentrations. This phenomenon was prevented by the protein kinase C inhibitor, bisindolylmaleimide. Moreover, N/OFQ‐induced desensitization did not affect mu opioid receptor responsiveness. Finally, it was observed in a similar preparation of rat cerebrocortical synaptosomes, although it was induced by higher N/OFQ concentrations than that used in the mouse. Together, these findings indicate that presynaptic NOP receptors inhibit 5‐hydroxytryptamine release in the mouse neocortex. Based on present and previous studies, we conclude that NOP receptors in the mouse are subtly different from the homologous receptor population in the rat, strengthening the view that there exist species differences in the pharmacology of central NOP receptors.