The glycine receptor (GlyR) is a member of the Cys-loop superfamily of ligand-gated ion channels and the major mediator of inhibitory neurotransmission in the spinal cord and brainstem. Many allosteric modulators affect the functioning of members of this superfamily, with some such as benzodiazepines showing great specificity and others such as zinc, alcohols, and volatile anesthetics acting on multiple members. To date, no potent and efficacious allosteric modulator acting specifically at the GlyR has been identified, hindering both experimental characterization of the receptor and development of GlyR-related therapeutics. We used phage display to identify novel peptides that specifically modulate GlyR function. Peptide D12-116 markedly enhanced GlyR currents at low micromolar concentrations but had no effects on the closely related ␥-aminobutyric acid type A receptors. This approach can readily be adapted for use with other channels that currently lack specific allosteric modulators.The glycine receptor (GlyR) 2 is a member of the Cys-loop superfamily of ligand-gated ion channels, including also the ␥-aminobutyric acid type A (GABA A ) and serotonin-3 receptors. They share a number of structural features, including ligand-binding sites in the extracellular N-terminal domain and a transmembrane domain consisting of four segments, with a large intracellular loop connecting segments 3 and 4. Individual channels consist of five subunits co-localized with the transmembrane domain 2 segment of each subunit lining the anionconducting pore (1, 2). Two classes of GlyR subunits have been identified: the ␣ subunits, of which there are four subtypes, and a single ␤ subunit (3). Most native GlyRs in adult animals consist of heteromeric ␣1␤ subunits, although homomeric ␣2 receptors are the predominant form found prenatally (4). GlyRs constitute the major inhibitory neurotransmitter receptor system in the brainstem and spinal cord (5), where they are thought to play a role in the modulation of pain signals and in the effects of volatile anesthetics (1). Some GlyR mutations result in the startle disorder hyperekplexia. GlyRs are also found throughout the brain, including the thalamus, hippocampus, and nucleus accumbens, where they were recently shown to be involved in the reinforcing properties of ethanol (6). The GlyR is only one of multiple ion channels and receptors thought to play a role in pain perception and alcohol and volatile anesthetic effects and in determining the state of neuronal excitability. The isolation of the role of the GlyR is hindered by the fact that, to date, no potent and efficacious allosteric modulator acting specifically at the GlyR has been identified. Phage display involves the expression of a random library of peptides on the coat proteins of bacteriophage. This method has long been used to identify peptides that can bind with high affinity to selected targets and to aid in identifying binding motifs (7). We combined phage display technology with standard electrophysiological testing to identi...