Abstract The respiratory system (RS: lung airways and alveolar parenchyma) and the urinary system (US:kidney, ureters and bladder) are both critical to homeostasis. The RS subserves gas exchange for cellular respiration and the US maintains fluid and electrolyte balance and supports elimination of toxic substances. Together, they regulate acid-base balance. Both arise via branching morphogenesis, are highly vascular, with a large endothelial/epithelial/neural interface, and eliminate unwanted substance to the outside environment. They are thus “barrier systems” that require extensive immune surveillance and are subject to environmental, infectious and, inflammatory injury, and fibrotic disrepair. Failure of either systems results in chronic debilitating diseases that are costly to manage. Improved understanding of both systems at a high-resolution, high content multiscale level and the parenchymal-extra cellular space interactions will provide remarkable insight into morphogenesis, homeostasis and injury/repair of the body’s principal barriers. Characterizing the identities of all the cell types and their spatial organization is the first step towards understanding the functions and malfunctions of these organs. In this project we propose to build spatial multi-omics maps of RS and US barrier organs at single-cell resolution, using a combination of highly efficient sequencing approach on dissociated cells, and highly multiplexed in situ mapping of RNA and proteins with fluorescent imaging. We plan to accomplish this goal with three specific aims in a 4-year funded period. Aim 1. We will establish an experimental and computation pipeline for generating and analyzing 3D multi- omics maps for human tissues. This pipeline will include single-nucleus RNA sequencing and chromatin accessibility mapping, highly multiplexed in situ imaging of RNA and proteins, and a computational framework to integrate different types of data to create coherent spatial maps of RNA, chromatin status and proteins. Aim 2. In one Organ Specific Project (OSP), we will create a Lung Airways and Prenchymal Map (LAPMAP), covering the entire intrathoracic human airway, as well as three critical niches of the human lung for both sexes. Aim 3. In a second OSP, we will create the Bladder Ureter and Kidney Maps (BUKMAP), covering human kidney and lower urinary tract.