A novel ligand for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-102), has been generated by combining in the N/OFQ-NH(2) sequence two chemical modifications, [Arg(14),Lys(15)] and [(pF)Phe(4)], that have been previously demonstrated to increase potency. In vitro, UFP-102 bound with high affinity to the human NOP receptor, showed at least 200-fold selectivity over classical opioid receptors, and mimicked N/OFQ effects in CHO(hNOP) cells, isolated tissues from various species, and mouse cortical synaptosomes releasing 5-hydroxytryptamine. UFP-102 showed similar maximal effects but higher potency (2- to 48-fold) relative to N/OFQ. The effects of UFP-102 were sensitive to NOP-selective antagonists J-113397 [(+/-)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] (pA(2) = 7.75-8.12) and UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2))(pA(2) = 6.91-7.33) but not to naloxone, and no longer observed in tissues taken from NOP receptor knockout mice (NOP(-/-)). In vivo, UFP-102 (0.01-0.3 nmol i.c.v.) mimicked the pronociceptive action of N/OFQ (0.1-10 nmol i.c.v.) in the mouse tail withdrawal assay, displaying higher potency and longer lasting effects. The action of UFP-102 was not apparent in NOP(-/-) mice. Similar results were obtained measuring locomotor activity in mice. In conscious rats, UFP-102 (0.05 nmol i.c.v.) produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate. These effects were comparable with those evoked by N/OFQ at 5 nmol. Collectively, these findings demonstrate that UFP-102 behaves as a highly potent and selective NOP receptor agonist that produces long-lasting effects in vivo.