The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is a non-opioid branch of the opioid receptor family implicated in several neurological and psychological disorders, such as pain, anxiety, depression, involuntary movement, addiction, seizure and dementia. Heterogeneity of NOP receptors has been proposed based on the findings of splicing variants and from binding and functional studies. We have previously reported that Ro 64-6198, a NOP receptor agonist, activated a subset, but not all, of N/OFQ-sensitive NOP receptors in midbrain ventrolateral periaqueductal grey (vlPAG). In this study, we found that a new NOP receptor ligand, (+)-5a Compound ((3aS, 6aR)-1-(cis-4-isopropylcyclohexyl)-5′-methyl-2′-phenylhexahydrospiro[piperidine-4,1′-pyrrolo[3, 4-c]pyrrole]), also activated a subset of NOP receptors in vlPAG neurons. (+)-5a Compound (0.1–30 μm) concentration-dependently activated G-protein-coupled inwardly-rectifying potassium (GIRK) channels mediated through the NOP receptors in about 35% of the recorded vlPAG neurons. (+)-5a Compound (EC50: 605 nm) was less potent (1/12) and efficacious (47%) than N/OFQ. In (+)-5a Compound-insensitive neurons, Ro 64-6198 was also ineffective, and vice versa, but N/OFQ activated GIRK channels through NOP receptors. In (+)-5a Compound-sensitive neurons, (+)-5a Compound precluded the effect of Ro 64-6198. Immunofluorecent and morphometric studies showed that most of the (+)-5a Compound-sensitive neurons were multipolar with intensive dendritic arborization and immunoreactive to glutamic acid decarboxylase-67. It is suggested that (+)-5a Compound activates a subset of NOP receptors, similar to the Ro 64-6198-sensitive subset, in the vlPAG neurons which are mostly GABAergic. These results further support the presence of functional heterogeneity of NOP receptors in the midbrain PAG.