PROJECT SUMMARY Distal (or small) airways including the terminal and respiratory bronchioles (TRB) are the sites of damage in several lung diseases. Pathologies of small airway lesions are poorly understood as the cellular composition and properties of TRB regions are yet to be fully characterized. Respiratory bronchioles (RB) are extremely small airways, about 200 µm in diameter. Importantly, the anatomy of mice – the most commonly used model organisms – is significantly different in these regions. Murine lungs lack RBs, and their terminal bronchioles open directly into alveoli at the bronchio-alveolar duct junction (BADJ). These challenges have resulted in RBs remaining as a black box of human lungs. We optimized microdissection protocols to enrich RBs from human lungs and generated single-cell transcriptomic maps. We identified seven novel cell populations in the TRB regions. Importantly, we show that these cells have no equivalent cell types in mice. All these data point towards the need to develop new models to effectively understand small airways in health and disease. We show here in our preliminary data that primary human stem cell cultures well capture the small airway biology. Further, we find that small airways of ferrets have similar cellular composition to those of humans suggesting that they can serve as a model to study small airways. Utilizing these two models coupled with single-cell transcriptomics we propose to address the following specific aims. 1) Delineate the terminal and respiratory bronchiolar epithelial lineage trajectories. 2) Study the cell-intrinsic transcriptional networks that maintain distal basal cell identity. At the conclusion of this study, we would have significantly advanced our understanding of human terminal and respiratory bronchioles. We will develop the first TRB-specific lineage tracing model in ferrets. Our study will determine the cell-intrinsic mechanisms that determine TRB stem cell identity and progeny. These results will assist in developing effective therapies for diseases involving small airways.