There are an insufficient number of donor organs available to meet the demand for lung transplantation. This issue could be addressed by regenerating functional tissue from diseased or damaged lungs that would otherwise be deemed unsuitable for transplant. Detergent-mediated whole-lung decellularization produces a three-dimensional natural scaffold that can be repopulated with various cell types. In this study, we investigated the decellularization and initial recellularization of diseased lungs using a rat model of monocrotaline-induced pulmonary hypertension (MCT-PHT). Decellularization of control and MCT-PHT Sprague-Dawley rat lungs was accomplished by treating the lungs with a combination of Triton X-100, sodium deoxycholate, NaCl, and DNase. The resulting acellular matrices were characterized by DNA quantification, Western blotting, immunohistochemistry, and proteomic analyses revealing that decellularization was able to remove cells while leaving the extracellular matrix (ECM) components and lung ultrastructure intact. Decellularization significantly reduced DNA content (∼30-fold in MCT-PHT lungs and ∼50-fold in the control lungs) and enriched ECM components (>60-fold in both the control and MCT-PHT lungs) while depleting cellular proteins. MicroCT visualization of MCT-PHT rat lungs indicated that the vasculature was narrowed as a result of MCT treatment, and this characteristic was unchanged by decellularization. Mean arterial vessel diameter of representative decellularized MCT-PHT and control scaffolds was estimated to be 0.152±0.134mm and 0.247±0.160mm, respectively. Decellularized MCT-PHT lung scaffolds supported attachment and survival of rat adipose-derived stem cells (rASCs), seeded into the airspace or the vasculature, for at least 2 weeks. The cells seeded in MCT-PHT lung scaffolds proliferated and underwent apoptosis similar to control scaffolds; however, the initial percentage of apoptotic cells was slightly higher in MCT-PHT lungs (2.79±2.03% vs. 1.05±1.02% of airway-seeded rASCs, and 4.47±1.21% vs. 2.66±0.10% of vascular seeded rASCs). The ECM of cell-seeded scaffolds showed no signs of degradation by the cells after 14 days in culture. These data suggest that diseased hypertensive lungs can be efficiently decellularized similar to control lungs and have the potential to be recellularized with mesenchymal stem cells with the ultimate goal of generating healthy, functional pulmonary tissue.