The N-methyl-D-aspartate receptor is a major target of alcohol action in the Central Nervous System. Alcohol influences the NMDA receptor channel gating and expression, but the underlying mechanisms have not been fully clarified. Recently we have demonstrated that clusters of five positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits form putative sites of alcohol action and interact to influence channel kinetics. Our previous studies have also shown that tryptophan substitution mutations at position GluN2A (M823) significantly increase at peak current density and the amount of increase was not attributable to an increase in receptor expression. In the present study, we investigated the effects of tryptophan mutations at putative sites of alcohol action on the effect of NMDA receptors surface expression using western blot in transiently-transfected HEK 293 cells. We found that GluN2A (M818W) significantly increased NMDA receptor surface membrane expression (p<0.05), but the corresponding position on the GluN2A subunit did not. Interestingly, previous studies have shown that GluN2A (M823W) increased ethanol IC50 value but GluN1 (M818W) did not. GluN2A (A825W) significantly altered the alcohol sensitivity, however, this mutant did not influence NMDA receptor surface expression. Our results indicate the fourth membrane-associated domains of GluN1 and GluN2A not only plays an important role in ethanol inhibition in NMDA receptors but also in surface expression.