Brain edema is a serious consequence of hemispheric stroke and traumatic brain injury and contributes significantly to patient mortality. In the present study, we measured water contents in hippocampal slices as an in vitro model of edema formation. Excitotoxic conditions induced by N-methyl-D-aspartate (NMDA, 300 microM), as well as ischemia induced by oxygen-glucose deprivation (OGD), caused cellular edema formation as indicated by an increase of slice water contents. In the presence of furosemide, an inhibitor of the Na,K,Cl-cotransporter, NMDA-induced edema were reduced by 64% while OGD-induced edema were unaffected. The same observation, i.e., reduction of excitotoxic edema formation but no effect on ischemia-induced edema, was made with chloride transport inhibitors such as DIDS and niflumic acid. Under ischemic conditions, modulation of GABAA receptors by bicuculline, a GABA antagonist, or by diazepam, a GABAergic agonist, did not significantly affect edema formation. Further experiments demonstrated that low chloride conditions prevented NMDA-induced, but not OGD-induced, water influx. Omission of calcium ions had no effect. Our results show that NMDA-induced edema formation is highly dependent on chloride influx as it was prevented by low-chloride conditions and by various compounds that interfere with chloride influx. In contrast, OGD-induced edema observed in brain slices was not affected by modulators of chloride fluxes. The results are discussed with reference to ionic changes occurring during tissue ischemia.