Hormone-activated nuclear receptors (NRs) control myriad cellular processes. The classical paradigm for hormone delivery is secretion from endocrine organs and blood-borne distribution to responding cells. However, many hormones can also be synthesized in the same tissues in which responding cells are found (paracrine signaling). In both endocrine and paracrine signaling, numerous factors affect hormone availability to target cell NRs, including hormone access to and sequestration by carrier proteins, transport across cell membranes, metabolism, and receptor availability. These factors can differ dramatically during development, between anatomical locations, and across cell types, and may cause highly variable responses to the same hormone signal. This has been difficult to study because current approaches are unable to quantify cell-intrinsic exposure to NR hormone ligands, precluding assessment of cell-specific hormone access and signaling. We have used the ligand-dependent interaction of the endogenous glucocorticoid (GC) receptor with chromatin as a biosensor that quantifies systemic access of GCs to cells within tissues at the single cell level, showing that tissues are buffered against circulating GCs. This approach also showed highly targeted paracrine GC signaling within the thymus, where GCs promote the positive selection of thymocytes with moderate affinity for self-antigens and the development of a safe and effective T-cell repertoire. We believe that this and complementary biosensor approaches will be useful to identify endocrine and paracrine target cells in situ and quantify their exposure to hormones regardless of the mode of delivery.