A fourth opioid receptor family was cloned and named after its endogenous ligand as nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor. We have characterized several NOP receptor ligands pharmacologically at native NOP receptors of ventrolateral periaqueductal gray (vlPAG) neurons by investigating their interactions with N/OFQ in activating G protein-coupled inwardly rectifying K+ (GIRK) channels. They are listed here: (1) [Phe1Psi(CH2-NH)Gly2]N/OFQ(1-13)NH2, which was claimed to be the first selective antagonist of NOP receptors, is a partial agonist of NOP receptors in vlPAG neurons. (2) [Nphe1]N/OFQ(1-13)NH2 is a pure, selective, and competitive peptide antagonist of NOP receptors (pA2 value = 6.6). (3) CompB (J-113397) is a potent and selective nonpeptide antagonist of NOP receptors (pA2 = 8.4). (4) Naloxone benzoylhydrazone is a competitive NOP receptor antagonist but also a noncompetitive mu-opioid receptor antagonist. (5) Ro 64-6198, though being developed as a potent nonpeptide NOP receptor agonist, affected only part of vlPAG neurons and acted as a weak NOP receptor agonist. In Ro 64-6198-unresponsive neurons, N/OFQ activated GIRK channels through NOP receptors. (6) Nocistatin, a functional antagonist of N/OFQ in the spinal cord, did not affect the effect of N/OFQ in most of the recorded neurons. Our functional studies of NOP receptor ligands at native brain NOP receptors reveal that some of them act differently from those at expressed receptors of cell cultures.