In vivo, pathological conditions such as ischemia and ischemia/reperfusion are known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Using an in vitro model of the BBB, consisting of porcine brain derived microvascular endothelial cells (BMEC), it was demonstrated that hypoxia-induced paracellular permeability across the cell monolayer was strongly aggravated by reoxygenation, which was prevented by catalase suggesting that H₂O₂ is the main mediator of the reoxygenation effect. Therefore, mechanisms leading to H₂O₂-induced permeability changes of BMECs were investigated by blockade of several intracellular signal transduction pathways. H₂O₂ increased the permeability of the BMEC monolayer, measured by the passage of [3H] inulin across the monolayer of primary cultures of BMECs, after 3 h incubation to 177 ± 12% (n = 10, p < 0.05 compared with untreated control cultures using unpaired Student's t test). Inhibition of phospholipase C (PLC) using U73122 (2.5 μM), inhibition of the intracellular Ca²⁺ release with TMB-8 (50 μM), and usage of the calmodulin antagonist W7 (40 μM) prevented H₂O_2-induced permeability changes during 3 h suggesting that H₂O₂ increases the intracellular Ca²⁺ concentration. Activation of the protein kinase C (PKC), phosphatidyl-inositol 3 kinase (PI3-K/Akt), guanylate cyclase (GC) and of the protein kinase G (PKG) are not involved in H₂O₂ induced hyperpermeability. But inhibition of the extracellular signal-regulated kinases (ERK1/ERK2 or p44/42 MAPK) using PD98059 (20 μM), but not of the p38 and of the c-jun NH2-terminal kinase (JNK), inhibited hyperpermeability by H₂O₂ completely. Western blot analysis confirmed the activation of the p44/42 MAP kinase. Corresponding to H₂O₂-induced permeability changes the phosphorylation of the p44/42 MAP kinase, which was increased by H₂O₂ to 248 ± 25% (n = 4, p < 0.05 compared with untreated control cultures) was inhibited by the specific MAP kinase inhibitor PD98059 and by TMB-8 and W7. Results suggest that H₂O₂-induced permeability changes are mediated by activation of PLC leading to the intracellular release of Ca²⁺ followed by activation of the p44/42 MAP kinase and paracellular permeability changes.