Chronic intermittent ethanol exposure (CIE) is associated with degeneration of hippocampal neurons. The present study used hippocampal cultures to examine the loss of NeuN immunoreactivity, a relaible marker or neuronal density, after 1, 2, or 3 cycles of 5 days EtOH exposure (50 mM), followed by a 24-hour period of EWD or continuous EtOH exposure. NeuN immunoreactivity was decreased by 13%, 19%, and 16% in the CA1, CA3, and dentate gyrus after 3 cycles of CIE respectively; thionine staining confirmed significant cellular losses within each hippocampal subregion. Two cycles of CIE in aged tissue cultures resulted in significant decreases in NeuN immunoreactivity in all hippocampal subregions; however continuous ethanol exposure or exposure to one cycle of CIE did not. Further, exposure to the N-Methyl-D-aspartate receptor (NMDAR) antagonist 2-amino-7-phosphonvaleric acid (APV) (30 uM) during periods of EWD attenuated the loss of NeuN in all hippocampal subregions, while exposure to APV (40 uM) prevented the loss of NeuN in the CA1 and dentate gyrus. These results suggest that the loss of mature neurons after CIE is associated with the overactivation on the NMDAR.