A recently identified novel peptide, orphanin FQ/nociceptin (OFQ/N), is an endogenous ligand for a unique member of the cloned opioid receptor family. Saturation studies in mouse brain membranes reveal curvilinear Scatchard plots with both a higher (KD 3.8 pM, Bmax 31.6 fmol/mg protein) and a lower (KD 896 pM, Bmax 233 fmol/mg protein) affinity site in brain tissue, compared to only a single site in transfected CHO cells (KD 36 pM). Competition studies confirm the high affinity of OFQ/N for this site, but shallow Hill slopes suggest heterogeneity. Traditional opioids have poor affinity for this receptor and OFQ/N and its fragments do not label traditional opioid receptors. In brain homogenates, both OFQ/N and OFQ/N(1-11) inhibit forskolin-stimulated camp accumulation with IC50 values of 1 nM or less, an action which is readily reversed by opioid antagonists. OFQ/N(1-7) shows little activity. Together, these studies suggest the presence of heterogeneous, functionally active OFQ/N receptors in mouse brain.