N-Methyl-D-aspartate (NMDA) receptors are a subtype of glutamate receptor that serve important functions at glutamatergic synapses in the brain. NMDA receptors are inhibited by concentrations of ethanol that are associated with intoxication and chronic exposure of neurons to ethanol enhances NMDA receptor function. The factors that underlie the acute inhibition of NMDA receptors by ethanol are not completely known, but ethanol sensitivity is influenced by receptor subunit composition. In this study, the effect of the regulatory subunit, NR3, on ethanol inhibition of NMDA receptors was examined. Recombinant NMDA receptors comprised of NR1 and NR2 (A-D) subunits were transiently transfected into HEK293 cells in the absence or presence of the NR3 subunit. In the absence of NR3, all NMDA receptor subunit combinations were inhibited by 100 mM ethanol. Co-expression of NR3 or an NR3-GFP fusion protein with NR1/NR2 (A-D) subunits did not alter the inhibitory effects of ethanol. In addition, the inhibition of NR1/NR2B receptors by the NR2B subunit-selective antagonist, ifenprodil, was not altered by co-expression of the NR3 subunit. Overall, these results suggest that the NR3A subunit is not a determinant of ethanol sensitivity in recombinant NMDA receptors.