Studies of nociceptin/orphanin FQ (NC) have been hampered by the paucity of available ligands with activity at the nociceptin receptor (NCR). In this study we have compared the agonist profile of NC and a novel NCR agonist, Ro65-6570, in a series of radioligand binding studies and effects on forskolin-stimulated cAMP formation in Chinese hamster ovary (CHO) cells expressing the recombinant human NCR (CHOhNCR). In addition, we report the effects of three antagonists, [Nphe1]NC(1-13)NH2, J-113397 and III-BTD, on these responses. In radioligand binding studies Ro65-6570, [Nphe1]NC(1-13)NH2, J-113397 and III-BTD displaced [3H]NC with similar pKi values (8.4-8.8). This compares with a pK(D) of 10.2 for NC in a direct saturation experiment. [Nphe1]NC(1-13)NH2 and J-113397 showed at least 100-fold selectivity over classical opioid receptors. Both NC and Ro65-6570 produced a concentration-dependent inhibition of cAMP formation with pEC50 values of 9.56+/-0.06 and 8.68+/-0.04, respectively. Maximum inhibition achieved was 100%. [Nphe1]NC(1-13)NH2, J-113397 and III-BTD produced a parallel rightward shift in the concentration-response curves to both NC and Ro65-6570 with pK(B) values of approximately 6.5, approximately 7.5 and approximately 7.7, respectively. Importantly, all three antagonists were devoid of residual agonist activity. Collectively, these data indicate the value of Ro65-6570, [Nphe1]NC(1-13)NH2, J-113397 and III-BTD in studies of the physiological role played by NC. However, due to the relatively poor selectivity of Ro65-6570 and III-BTD caution should be exercised when using tissues that co-express micro-opioid receptors.