To determine the cellular targets for glucocorticoid (GC) action, we have generated mice in which a green fluorescent protein-glucocorticoid receptor (GFP-GR) fusion gene is knocked into the endogenous GR locus. We found that GFP-GR function is indistinguishable from endogenous GR on both a cellular and systemic level. Furthermore, the green fluorescence intensity of the GFP-GR protein is proportional to its expression, allowing quantitation of GR expression in single living cells. We initiated our analysis of GR regulation in the thymus. Using multicolor flow cytometry, we found that GR expression is uniform among embryonic thymocyte subpopulations, but gradually "matures" over a three-week period after birth. In the adult, analysis of GFP-GR expression on RAG2-/- and HY T cell receptor (TCR) transgenic genetic backgrounds, showed that GR is induced to high levels in immature CD25+ CD4- CD8- thymocytes and down-regulated by activation of the pre-TCR during positive but not negative selection. Additionally, relative GR expression is dissociated from GC-induced apoptosis in vivo. These results implicate pre-TCR signaling as a mechanism for GR down-regulation and separate receptor abundance from susceptibility to apoptosis across thymocyte populations.