Ethanol is a potent inhibitor of the function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor in various neuronal preparations. In primary cultures of cerebellar granule cells, ethanol was suggested to interact with the glycine co-agonist site of the receptor by a mechanism involving protein kinase C. In the present study, the interaction of ethanol with various sites on the NMDA receptor was examined in primary cultures of cerebral cortical cells from embryonic rats. NMDA receptor function was determined by measuring increases in intracellular Ca2+ with fura-2 fluorescence. Ethanol inhibited the function of the NMDA receptor in cerebral cortical cells, but in contrast to the results in cerebellar granule cells, phorbol ester treatment did not inhibit the NMDA response, and ethanol did not alter the effect of glycine on NMDA receptor function. Ethanol also did not affect inhibition of the NMDA response by Mg2+ or dizocilpine. The results support the hypothesis that the mechanism of ethanol inhibition of NMDA receptor function can vary in neurons from different brain regions.