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Recent reports have identified an endogenous peptide ligand for the opioid receptor-like (ORL1) receptor. In the present study, ORL1 peptide-stimulated [35S]GTPγS binding was assessed in rat cortical membranes and brain sections to localize ORL1 receptor-activated G-proteins. In membrane assays, with 20 γM GDP, ORL1 peptide stimulated [35S]GTPγS binding by approximately twofold with an ED50 value of 20 nM. ORL1 peptide- stimulated [35S]GTPγS binding was unaffected by opioid or other G-protein-coupled receptor antagonists. In brain sections, ORL1 peptide-stimulated [35S]GTPγS binding was identified in regions including cortex, amygdala, hypothalamus, thalamus and brain stem. The anatomical distribution of ORL1 peptide-stimulated [35S]GTPγS binding suggests its involvement in cognition, emotion and homeostasis.