The mechanism by which ethanol inhibits the function of the NMDA subtype of glutamate receptor has not been elucidated. One possibility that has been suggested is that NMDA receptor subunit composition influences the sensitivity of the receptor to ethanol. We have taken advantage of developmental changes in subunit composition of the NMDA receptor in cultured neurons to examine possible changes in the effect of ethanol. We found an increase in expression of the NR2A subunit, and a decrease in expression of the NR2B subunit of the NMDA receptor in primary cultures of cerebellar granule neurons over time in culture, with no significant change in NR1 expression. This change in NR2 subunit expression was associated with the expected changes in functional properties of the NMDA receptor (measured as the NMDA-induced increase in intracellular Ca2+), i.e., ifenprodil sensitivity and glycine potency were higher when there was a relatively greater proportion of NR2B in the cultured neurons. However, the potency of ethanol to inhibit NMDA receptor function was lower when there was a greater proportion of NR2B subunits. Previous studies showed that ethanol inhibition of NMDA receptor function in cerebellar granule neurons resulted from an ethanol-induced decrease in potency of the co-agonist, glycine, and that this effect of ethanol was blocked by inhibitors of protein kinase C. Our current results suggest that the lower potency of ethanol to inhibit the response of NMDA receptors when cerebellar granule neurons are expressing a greater proportion of NR2B subunits is a result of the higher affinity of the NMDA receptors for endogenous levels of glycine at this point in time.