The hexapeptide ac-RYYRWK-NH2 has been described as a potent partial agonist at the nociceptin (NC)/orphanin FQ receptor which has no affinity for mu-, kappa- or delta-opioid receptors. However, it is not clear whether ac-RYYRWK-NH2 is truly selective for the NC receptor, and ac-RYYRWK-NH2 has therefore been radiolabelled and characterised in receptor-binding experiments. Saturation experiments with [3H]ac-RYYRWK-NH2 binding to rat cortical membranes revealed a single high affinity site for [3H]ac-RYYRWK-NH2 (Kd=0.071 +/- 0.018 nM; Bmax=22+/-2 fmol/mg protein). Uncoupling of the G-proteins resulted in a significant 45% increase in Kd and no change in Bmax. [3H]ac-RYYRWK-NH2 binding to rat cortical membranes or to membranes from baby hamster kidney cells expressing human orphan opioid receptor-like (ORL1) was displaced by NC and ac-RYYRWK-NH2 to the same extent. The following rank order of potency was observed: ac-RYYRWK-NH2 > [Tyr14]NC-OH = NC-OH = NC-NH2 > NC, H-(1-13)-NH2 > NC(1-12)-NH2 >> NC(1-11)-NH2 and, thus, displayed a typical NC receptor pharmacology. Novel cyclic analogues of ac-RYYRWK-NH2 were prepared but these structures were much less active when compared to ac-RYYRWK-NH2. In vitro receptor autoradiography with [3H]ac-RYYRWK-NH2 to rat brain sections revealed high levels of binding in the cerebral cortex, amygdala, hypothalamus and superior colliculus, but low levels in the cerebellum and striatum. Overall, the regional distribution was very similar to that of [3H]NC. Ac-RYYRWK-NH2 seems indeed to be selective for the NC receptor and [3H]ac-RYYRWK-NH2 is a novel radioligand which may be useful for further exploring the pharmacology and receptor-ligand interaction of the NC receptor.