The relationship between binding affinity and functional activity of nociceptin/orphanin FQ binding was studied in brain membranes, membranes of Chinese hamster ovary cells transfected with ORL1, and intact CHO-ORL1 cells. Binding affinities were compared with potency for the stimulation of [35S]GTP gamma S binding in cell membranes, and inhibition of forskolin-stimulated cAMP accumulation in intact cells. Binding was conducted with [3H]14-Tyr-nociceptin, and in brain or cell membranes the affinity was found to be 50-100 pM. The binding of [3H]14-Tyr-nociceptin was found to be regulated by Na+ and GTP, as expected for an opioid-like receptor. In intact cells, saturation produced a curvilinear Scatchard Plot. Non-linear analysis indicated two states of the receptor, with the vast majority of binding being to a low affinity state of approximately 8 nM. This low affinity component is consistent with the lower potency derived from the inhibition of cAMP accumulation, stimulation of [35S]GTP gamma S binding, and other functional assays.