Endothelial (EC) junctions determine vascular barrier properties and are subject to transient opening to allow liquid flux from blood to tissue. Although EC junctions open in the presence of permeability enhancing factors, including oxidants, the mechanisms by which they reseal remain inadequately understood. To model opening and resealing of EC junctions in the presence of an oxidant, we quantified H₂O₂-induced trans-endothelial resistance (TER) in monolayers of rat lung microvascular EC. During a 30-min exposure, H₂O₂ (100 µM) decreased TER for an initial ~10 min, indicating junctional opening. Subsequently, despite continuous presence of H₂O₂ TER recovered to baseline, indicating the activation of junctional resealing mechanisms. These bi-modal TER transients matched the time course of loss and then gain of E-cadherin at EC junctions. The timing of the TER decrease matched the onset of focal adhesion formation, while f-actin increase at the cell periphery occurred with a time course that complemented the recovery of perpheral E-cadherin. In monolayers expressing a focal adhesion kinase (FAK) mutant (del-FAK) that inhibits FAK activity, the initial H₂O₂-induced junctional opening was present, although the subsequent junctional recovery was blocked. Expression of transfected E-cadherin was evident at the cell periphery of wild type, but not in del-FAK-expressing ECs. E-cadherin overexpression in del-FAK-expressing ECs failed to effect major rescue of the junctional resealing response. These findings indicate that in oxidant-induced EC junction opening, FAK plays a critical role in remodeling the adherens junction to reseal the barrier.