Re-expansion of a collapsed lung increases the microvascular permeability and causes re-expansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its targets Rho-kinase (ROCK) regulate endothelial permeability, though their roles in re-expansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue/plasma (T/P) and broncho-alveolar lavage (BAL) fluid/plasma (B/P) ratio with ¹²⁵I-albumin. After intratracheal instillation of ¹²⁵I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of ¹²⁵I-albumin in plasma/total amount of instilled ¹²⁵I-albumin. T/P, B/P and PL index were significantly increased in the re-expanded lung. These increases were attenuated by pre-treatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the re-expanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H₂O₂-induced increase in permeability, and mitigated the morphologic changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with re-expansion pulmonary edema.