Underphysiologic conditions, brain intracellular pH (pH(i)) is maintained at 7.03. Rebound brain intracellular alkalosis has been observed in experimental models and adult stroke after hypoxia/ischemia (HI). In term infants with neonatal encephalopathy (NE), an association exists between the magnitude of brain alkalosis and neurodevelopmental outcome, and there is increasing evidence to suggest that alkalosis may be deleterious to cell survival. Activation of the Na(+)/H(+) exchanger (NHE) is thought to be responsible for the rapid normalization of pH(i) and rebound alkalosis after reperfusion. We hypothesized that N-methyl-isobutyl-amiloride (MIA), an inhibitor of the NHE, would reduce brain injury in a model of neonatal HI. Seven-day-old mice underwent left carotid artery occlusion followed by exposure to 8% oxygen for 30 min (moderate insult) or 1 h (severe insult). Animals received MIA or saline 8 hourly starting 30 min before HI. Outcome was determined at 48 h by measuring viable tissue in the injured hemisphere (severe insult) or injury score and TUNEL staining (moderate insult). After the severe insult, MIA had a significant neuroprotective effect increasing forebrain tissue survival from 44% to 67%. After the moderate insult, damage was localized to the hippocampus where treatment resulted in a significant reduction in injury score and in TUNEL-positive cells. MIA was also shown to have a significant overall neuroprotective effect based on injury score after the moderate insult. Amiloride analogues are neuroprotective when commenced before HI in a mouse model.