Nociceptin/orphanin FQ (N/OFQ) and N/OFQ peptide (NOP) receptors are implicated in many physiological functions including pain regulation. This study quantitatively investigated the interaction of a novel NOP receptor antagonist, UFP-101 ([Nphe 1 ,Arg 14 ,Lys 15 ]N/OFQ-NH 2 ), with N/OFQ in the ventrolateral periaqueductal gray, a crucial midbrain area for pain regulation. N/OFQ concentration-dependently activated G-protein coupled inwardly rectifying K + (GIRK) channels in ventrolateral neurons of periaqueductal gray slices. UFP-101 antagonized N/OFQ-induced GIRK channel activation in a concentration-dependent manner and produced a parallel shift of the concentration–response curve of N/OFQ. The pA 2 value estimated from Schild plot is 6.92±0.06. At concentrations up to 1 μM, UFP-101 had no effect on membrane current per se and did not affect the GIRK current activated by [d-Ala 2 , N-Me-Phe 4 , Gly-ol 5 ]-enkephalin, a μ-opioid receptor agonist. It is concluded that UFP-101 is a potent and competitive peptide antagonist of NOP receptors that mediate GIRK channel activation in ventrolateral periaqueductal gray neurons.