Abstract Intracerebral microdialysis was employed in awake freely moving rats to investigate the effects of nociceptin/orphanin FQ receptor ligands on glutamate extracellular levels in the substantia nigra pars reticulata. Nociceptin/orphanin FQ, ineffective at 0.1 μM, induced a prolonged stimulation of nigral glutamate levels at 1 and 10 μM (mean effect of 137±9 and 167±13%, respectively, of basal values). These effects were prevented by the novel nociceptin/orphanin FQ receptor antagonist [Nphe 1]nociceptin/orphanin FQ(1-13)NH 2 (100 and 300 μM, respectively) but not by the non-selective opioid receptor antagonist naloxone (10 μM). [Nphe 1]nociceptin/orphanin FQ(1-13)NH 2 (100 μM) inhibited by itself glutamate outflow (maximal reduction to 71±4%) while naloxone was ineffective. The nociceptin/orphanin FQ receptor ligand [Phe 1ψ(CH 2-NH)Gly 2]nociceptin/orphanin FQ(1-13)NH 2 also facilitated glutamate outflow at 10 μM (mean effect of 145±10%). Intranigral perfusion with tetrodotoxin (1 μM) or with the dopamine D 2 receptor antagonist raclopride (1 μM), failed to affect basal glutamate output and prevented the facilitatory effect of nociceptin/orphanin FQ (10 μM). However, perfusion with the GABA A receptor antagonist bicuculline (10 μM) increased local glutamate extracellular levels by itself and attenuated the effect of the peptide. Our data suggest that nociceptin/orphanin FQ increases glutamate extracellular levels in the substantia nigra pars reticulata via activation of nociceptin/orphanin FQ receptors located on non-glutamatergic, possibly dopaminergic and GABAergic, neuronal elements.