Abstract: Acidosis is a universal response of tissue to ischemia. In the brain, severe acidosis has been linked to worsening of cerebral infarction. However, milder acidosis can have protective effects. As part of our investigations of the therapeutic window in our neuronal tissue culture model of ischemia, we investigated the effects of acidosis during recovery from brief simulated ischemia. Ischemic conditions were simulated in dissociated cortical cultures by metabolic inhibition with potassium cyanide to block oxidative metabolism and 2-deoxyglucose to block glycolysis. Lowering the extracellular pH (pHe) to 6.2 during metabolic inhibition had no effect on injury, as measured by lactate dehydrogenase release from cultures after 24 h of recovery. Lowering the pHe during the first hour of recovery, in contrast, had profound protective effects. When the duration of metabolic inhibition was lengthened to 30 min, most of the protective effects of the NMDA receptor antagonist MK-801 were lost. However, the protective effects of acidosis were unchanged. This suggested that the protective effects of extracellular acidosis could be due to more than blockade of NMDA receptors. Intracellular acidosis might be responsible. To test this, recovery of intracellular pH (pHi) was slowed by incubation with blockers of Na+/H+ exchangers at normal pHe. The two compounds tested, dimethylamiloride and harmaline, had protective effects when present during recovery from metabolic inhibition. Measurements of pHi confirmed that the blockers slowed recovery from intracellular acidosis; more rapid pHi recovery was correlated with injury. The protective effects of acidosis could be reversed by brief incubation with the protonophore monensin, which rapidly normalized pHi. These results are the first demonstration of the protective effects of blocking Na+/H+ exchange in a model of cerebral ischemia. The protective effects of acidosis appear to arise either from suppressing pH-sensitive mechanisms of injury or from blocking sodium entry due to Na+/H+ exchange.