Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). Binding studies have relied on [leucyl-(3)H]N/OFQ, but as this is an agonist G-protein coupling will affect affinity. In this paper, we describe a new [(3)H]labeled NOP antagonist, [Nphe(1),4'-(3)H-Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) ([(3)H]UFP-101). We have characterized [(3)H]UFP-101 at recombinant human NOP expressed in Chinese hamster ovary cells (CHO(hNOP)) and native rat NOP in cerebrocortex. Radioligand saturation and competition studies were performed on membranes, and [(3)H]UFP-101 (antagonist) was compared with [(3)H]N/OFQ (agonist). The effects of GTPgammaS (10 microM) and Na(+) were investigated alone and in combination in competition experiments with both radioligands. In CHO(hNOP), B (max), and pK (D), values were 561 and 580 fmol mg protein(-1) and 9.97 and 10.19 for [(3)H]UFP-101 and [leucyl-(3)H]N/OFQ, respectively. In rat cerebrocortex B (max) and pK (D), values were 65 and 88 fmol mg protein(-1) and 10.12 and 10.34 for [(3)H]UFP-101 and [leucyl-(3)H]N/OFQ. The binding of both radioligands was displaced by a range of peptide and non-peptide NOP ligands at both isoforms with good correlation (r (2) 0.92 in Rat and 0.97 in CHO(hNOP)). Naloxone was inactive. The binding of both radioligands was Na(+)-dependent with [(3)H]UFP-101 being more sensitive (IC(50) approximately20 mM). Unlike the agonist [leucyl-(3)H]N/OFQ, the antagonist [(3)H]UFP-101 was unaffected by GTPgammaS. [(3)H]UFP-101 binds to human and rat NOP with high affinity and good agreement with standard [leucyl-(3)H]N/OFQ in competition experiments. In addition, the binding of [(3)H]UFP-101 is unaffected by GTPgammaS. This radioligand will be useful to further characterize NOP in a range of binding paradigms.