Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP) which is yet to be functionally characterized in dog brain. Ligand binding data reports low NOP density (29 fmol mg - 1 protein) in dog. In this study using dog brain membranes, we have examined the effects of N/OFQ on [leucyl- 3 H]N/OFQ(1-17)OH ([leucyl- 3 H]N/OFQ) binding in the presence and absence of 120 mM NaCl and 100 μM GTPγS. Data from standard [ 3 5 S]GTPγS binding and immunoprecipitation (G α i 1 - 3 ) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHO h N O P ) cells. N/OFQ displaced [leucyl- 3 H]N/OFQ binding with pK i and slope values of 9.62+/-0.07 and 0.38+/-0.05, respectively. Addition of NaCl/GTPγS produced a steepening (slope 0.95+/-0.06, n=3) of the curve. N/OFQ stimulated [ 3 5 S]GTPγS binding with pEC 5 0 and E m a x values of 8.21+/-0.17 and 1.17+/-0.01, respectively (in CHO h N O P , pEC 5 0 and E m a x values were 8.47+/-0.01 and 7.01+/-0.63). N/OFQ stimulated [ 3 5 S]GTPγS binding in dog and CHO h N O P cell membranes could be immunoprecipitated with an anti-G α i 1 - 3 antibody, indicating coupling to a pertussis toxin (PTx)-sensitive G-protein. N/OFQ actions were competitively antagonized by the selective NOP antagonists, 100 nM J-113397, 1 μM [Nphe 1 ]N/OFQ(1-13)NH 2 and 1 μM [Phe 1 Ψ(CH 2 -NH)Gly 2 ]N/OFQ(1-13)NH 2 (partial agonist) yielding pK B values of 8.58+/-0.21, 7.06+/-0.59 and 7.32+/-0.41, respectively (in CHO h N O P , a pK B for J-113397 of 8.33+/-0.02 was obtained). Despite relatively low receptor density, we were able to detect functional activity of native dog NOP, with pharmacology consistent with reports for other species.