We have utilized a hippocampal brain slice explant system to assess cellular and synaptic mechanisms underlying the expression of alcohol withdrawal hyperexcitability. Previously, we observed a role for NMDA receptors in the expression of electrographic seizures (EGS) observed immediately upon withdrawal from chronic ethanol exposure in this system. One possible cellular mechanism responsible for these prior results involves NMDAR-mediated neurotoxicity, which was assessed in the present study. Explants were exposed to 35 or 75 mM ethanol for 6 or 12 days and incubated with propidium iodide (PI) to label non-viable cells and then imaged digitally. PI labeling was significantly reduced (36% of control levels) following chronic ethanol exposure (75 mM). When tested following ethanol withdrawal, PI labeling remained significantly reduced in the 75 mM exposed group. We next assessed the effect of an NMDA challenge 24 h following withdrawal. The 35 mM and 75 mM ethanol exposed groups displayed significant 6-fold and 13-fold NMDAR-mediated increases in PI labeling respectively; control explants displayed a 3-fold increase. These data suggest that chronic ethanol exposure prior to withdrawal has a minor neuroprotective effect that slightly diminishes within 24 h of ethanol withdrawal. Furthermore, the data indicate that direct NMDAR activation is required for induction of ethanol withdrawal neurotoxicity.