N-Methyl-D-aspartate (NMDA) receptors are important targets for drugs of abuse such as ethanol, toluene, and ketamine. Ligand-gated ion channels assembled from the NR1 and NR3 subunits have functional and pharmacological properties that are distinct from those of conventional NMDA receptors containing NR2 subunits. In the present study we used voltageclamp electrophysiology to characterize excitatory glycine-activated receptors assembled from NR1, NR3A, and NR3B subunits expressed in human embryonic kidney (HEK) 293 cells. These glycine-activated receptors were not stimulated by glutamate or kainic acid and were resistant to magnesium block. A wide variety of NMDA receptor antagonists including D-2-amino-5-phosphonovaleric acid, ifenprodil, memantine, (5R,10S)-(ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or acamprosate did not inhibit glycine-activated NR1/NR3A/NR3B receptors. Likewise, these receptors were not affected by antagonists of inhibitory glycine receptors or glycine transporters. The NMDA receptor glycine site agonist, D-serine, partially activated NR1/ NR3A/NR3B receptors, whereas the antagonist, 5,7-dichlorokynurenic acid, inhibited receptor currents. Conversely, the antagonist, 7-chlorokynurenic acid, and the partial agonist, R-(ϩ)-3-amino-1-hydroxy-2-pyrrolidinone (HA-966), potentiated glycine-stimulated currents of these receptors. NR1/ NR3A/NR3B receptor currents were inhibited by 10 to 21% by ethanol and toluene but were relatively insensitive to ketamine. Ethanol inhibition was enhanced in receptors expressing the NR1(L819A) mutant, whereas those containing NR1(F639A) or NR1(M813A) showed no change relative to the wild-type NR1. The results of this study indicate that coexpression of NR1, NR3A, and NR3B subunits in HEK 293 cells results in glycineactivated receptors with novel functional and pharmacological properties.N-Methyl-D-aspartate (NMDA) receptors are glutamateactivated ion channels that contain NR1 and NR2 subunits. A third class of NMDA receptor subunits has also been reported and consists of the NR3A and NR3B subunits (Ciabarra et al., 1995;Sun et al., 1998). NR3 subunits share structural features with the NR1 subunit, including the S1 and S2 agonist binding domains and some sequence similarity across transmembrane domains (Chatterton et al., 2002). Coexpression of the NR3A or NR3B subunits with NR1 and NR2 in recombinant expression systems decreases NMDA receptor current (Chatterton et al., 2002) and reduces calcium permeability (Matsuda et al., 2002). In NR3A-deficient mice, NMDA receptor currents are significantly enhanced (Das et al., 1998), suggesting that these subunits may be important modulators of NMDA receptor function.As for NR1 subunits, NR3 subunits do not form functional channels when expressed alone. However, coexpression of NR1 and NR3 subunits in oocytes has been reported to produce functional channels that are gated by glycine but not glutamate (Chatterton et al., 2002; but see Smothers and Woodward, 2003). In...