Summary, Organ Specific Project Human skin and its appendages comprise the largest organ in the body. In addition to its barrier function, the skin is involved in tasks related to maintaining homeostatic control such as temperature regulation, maintaining mechano-structural integrity and resilience, and serving as a reservoir for immune and other cell types that are critical for defense and wound repair. Mechanistic studies have shown that deleterious changes to these functions stem from combinations of both intrinsic and extrinsic factors. These factors are then influenced by age, sex, and race. As humans age, nearly all skin functions are affected. These include the loss of structural integrity and resilience, impaired self-renewal capabilities, poor wound repair and scarring, poor temperature regulation from deficiencies in eccrine sweat glands, decreased hormone production, impaired pigmentation and susceptibility to environmental damage, and increased inflammation and senescence. These changes lead to skin fragility and disease, pointing to the increased need for enhanced understanding of the skin microenvironment and its age-, sex-, and race-associated changes. In addition to the age-, sex-, and race- associated mechano-structural changes to the architecture of the ECM, the cellular ecosystem within the skin changes significantly over the lifespan. Within the 3D skin microenvironment, changes in ECM architecture, cell- ECM interactions, abundance of cell types/subpopulations, their underlying molecular activity/functional gene expression patterns, and heterogeneity across different body locations are poorly understood. We propose six Aims to map the normal, non-diseased skin as a function of age, race, sex, and location on the body. The first two Aims of the Project will produce 3D reference architectural maps of skin as a function of age, sex, race, and skin location, while the next three Aims will produce associated 3D reference cellular maps. Architectural mapping (Aims 1-2) will assess the 3D molecular composition and morphology/topology of the major multi-cellular structures of skin, including epidermis, dermis, hair follicles, apocrine and eccrine glands, blood vessels, and nerves. Aims 1-2 will also measure morphology and degree of alignment of the ECM of skin in 3D. Cellular mapping (Aims 3-5) will produce exhaustive spatially resolved cellular and molecular profiles of hair follicles, epidermis, and dermis, as well as large-volume 3D “immune cell maps”, “stromal cell maps”, and “stem cell maps” of the entire skin. High-content integration (Aim 6) will produce a user-friendly software that provides non-experts with the ability to interrogate context-dependent reference maps integrating both architectural and cellular compartments.