H+ extrusion is important for sustained NADPH oxidase activation after "respiratory" burst in macrophage/microglia activation. In this study, we investigated the role of Na+/H+ exchanger isoform 1 (NHE-1) in activation of microglia after lipopolysaccharide (LPS) or oxygen and glucose deprivation and reoxygenation (OGD/REOX) exposure. NHE-1 functioned in maintaining basal pHi of immortalized M4T.4 microglia or mouse primary microglia. Pharmacological inhibition of NHE-1 activity with the potent inhibitor cariporide [HOE 642 (4-isopropyl-3-methylsulfonyl-benzoyl- guanidine-methanesulfonate)] abolished pHi regulation in microglia under basal conditions. Activation of microglia either by LPS, phorbol myristate acetate, or OGD/REOX accelerated pHi regulation and caused pH i elevation, which was accompanied with an increase in [Na +]i and [Ca2+]i as well as production of superoxide anion and cytokines. Interestingly, inhibition of NHE-1 not only abolished pHi regulation but also reduced production of superoxide anion as well as expression of cytokines and inducible nitric oxide synthase. Together, these results reveal that there was a concurrent activation of NHE-1 in microglia in response to proinflammatory stimuli. The study suggests that NHE-1 functions to maintain microglial pHi homeostasis allowing for sustained NADPH oxidase function and "respiratory" burst.