Inhibitory glycine receptors (GlyRs) in the mammalian cortex probably contribute to brain development and to maintaining tonic inhibition. Given their presence throughout the cortex, their modulation likely has important physiological consequences. Although benzodiazepines potentiate γ-aminobutyric acidA, receptors (GABAARs), they may also modulate GlyRs because binding studies initially suggested that they act at GlyRs. Furthermore, their diminished ability to potentiate neonatal GABAARs suggests that they may exert their beneficial clinical effects at another site in the developing brain. Therefore we examined the effect of benzodiazepines on whole cell currents mediated by GlyRs in cultured embryonic mouse hippocampal neurons. First, we determined the GlyR subunit composition in this preparation. Glycine, β-alanine, and taurine activate strychnine-sensitive chloride currents in a dose-dependent manner. Maximal concentrations of the three agonists produce equal, nonadditive responses as expected of full agonists. The pharmacological properties of the GlyR currents including their pattern of modulation by picrotoxinin, picrotin, and tropisetron indicate that GlyRs consist of α2β heteromers and α2 homomers. Reverse transcriptase polymerase chain reaction (RTPCR) studies confirmed the presence of α2 and β subunits. Second, we found that micromolar concentrations of some benzodiazepines, including chlordiazepoxide and nitrazepam, inhibit GlyR currents. Nitrazepam inhibition of GlyRs is noncompetitive, is not voltage dependent, and does not reflect enhanced desensitization. Thus benzodiazepines allosterically inhibit α2-containing GlyRs in embryonic mouse hippocampal neurons via a "low "-affinity site.