We found that stretching Type I rat alveolar epithelial cell (RAEC) monolayers at magnitudes that correspond to high tidal-volume mechanical ventilation results in the production of reactive oxygen species, including nitric oxide and superoxide. Scavenging superoxide with Tiron eliminated the stretch-induced increase in cell monolayer permeability, and similar results were reported for rats ventilated at large tidal volumes, suggesting that oxidative stress plays an important role in barrier impairment in ventilator-induced lung injury associated with large stretch and tidal volumes. In this communication we show that mechanisms that involve oxidative injury are also present in a novel precision cut lung slices (PCLS) model under identical mechanical loads. PCLSs from healthy rats were stretched cyclically to 37% change in surface area for 1 hour. Superoxide was visualized using MitoSOX. To evaluate functional relationships, in separate stretch studies superoxide was scavenged using Tiron or mito-Tempo. PCLS and RAEC permeability was assessed as tight junction (TJ) protein (occludin, claudin-4 and claudin-7) dissociation from zona occludins-1 (ZO-1) via co-immunoprecipitation and Western blot, after 1 h (PCLS) or 10 min (RAEC) of stretch. Superoxide was increased significantly in PCLS, and Tiron and mito-Tempo dramatically attenuated the response, preventing claudin-4 and claudin-7 dissociation from ZO-1. Using a novel PCLS model for ventilator-induced lung injury studies, we have shown that uniform, biaxial, cyclic stretch generates ROS in the slices, and that superoxide scavenging that can protect the lung tissue under stretch conditions. We conclude that PCLS offer a valuable platform for investigating antioxidant treatments to prevent ventilation-induced lung injury.