Blood-brain barrier (BBB) Na transporters are essential for brain water and electrolyte homeostasis. However, they also contribute to edema formation during the early hours of ischemic stroke through increased transport of Na from blood into brain across an intact BBB. We have shown previously that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia, aglycemia and arginine vasopressin (AVP), and that inhibition of the cotransporter by intravenous bumetanide significantly reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of stroke. More recently we have found evidence that intravenous cariporide (HOE-642), a highly potent Na/H exchange inhibitor, also reduces brain edema after MCAO. The present study was conducted to investigate which Na/H exchanger (NHE) protein isoforms are present in BBB endothelial cells and to evaluate the effects of ischemic factors on BBB NHE activity. Western blot analysis of bovine cerebral microvascular endothelial cells (CMEC) and immunoelectron microscopy of perfusion fixed rat brain revealed that both NHE1 and NHE2 isoforms are present in BBB endothelial cells. Hypoxia (2% O₂, 30 min), aglycemia (30 min) or AVP (1-200 nM, 5 min) all significantly increased CMEC NHE activity, assessed as Na-dependent, HOE-642-sensitive H⁺ flux using microspectrofluorometry and the pH-sensitive dye BCECF. We found that AVP stimulation of CMEC NHE activity is dependent on intracellular [Ca] and is blocked by V₁ but not V₂ VP antagonists. Our findings support the hypothesis that a BBB Na/H exchanger, possibly NHE1 and/or NHE2, is stimulated during ischemia to participate in cerebral edema formation.