We measured stretch-induced changes in transepithelial permeability in vitro to uncharged tracers 1.5–5.5 Å in radius to identify a critical stretch threshold associated with failure of the alveolar epithelial transport barrier. Cultured alveolar epithelial cells were subjected to a uniform cyclic (0.25 Hz) biaxial 12, 25, or 37% change in surface area (ΔSA) for 1 h. Additional cells served as unstretched controls. Only 37% ΔSA (100% total lung capacity) produced a significant increase in transepithelial tracer permeability, with the largest increases for bigger tracers. Using the permeability data, we modeled the epithelial permeability in each group as a population of small pores punctuated by occasional large pores. After 37% ΔSA, increases in paracellular transport were correlated with increases in the radii of both pore populations. Inhibition of protein kinase C and tyrosine kinase activity during stretch did not affect the permeability of stretched cells. In contrast, chelating intracellular calcium and/or stabilizing F-actin during 37% ΔSA stretch reduced but did not eliminate the stretch-induced increase in paracellular permeability. These results provide the first in vitro evidence that large magnitudes of stretch increase paracellular transport of micromolecules across the alveolar epithelium, partially mediated by intracellular signaling pathways. Our monolayer data are supported by whole lung permeability results, which also show an increase in alveolar permeability at high inflation volumes (20 ml/kg) at the same rate for both healthy and septic lungs.