Members of the ligand-induced nuclear receptor superfamily are medically important regulators of cellular activity. Hormones initiate a receptor activation process leading to receptor redistribution and binding to specific DNA recognition sites in the promoter regions of their target genes. Using microscopy and fluorescent protein chimeras of nuclear receptors, we pioneered studies establishing that ligand binding regulates the subcellular targeting of glucocorticoid (GR), vitamin D (VDR), and retinoid X receptors (RXR). We have continued to use advanced microscopy techniques to address the roles of receptor trafficking in hormone actions. We demonstrated that heterodimerization with RXR has a profound effect on VDR subcellular distribution and that this effect is physiologically important. When expressed separately, the steady-state distribution of the YFP-RXR was more nuclear than the GFP-VDR. Experiments measuring fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that both VDR and RXR constantly shuttle between the cytoplasm and the nucleus and that RXR shuttles at a slower rate than VDR. Coexpression of RXR-BFP with GFP-VDR promoted nuclear accumulation of the later by influencing both nuclear import and retention. RXR-BFP also promoted hormone-dependent nuclear accumulation of a nuclear localization signal (NLS) mutant receptor (nlsGFP-VDR), and rescued its transcriptional activity. Heterodimerization mutant RXR failed to alter GFP-VDR and nlsGFP-VDR distribution or activity. We identified the NLS of the RXR and found that the mutant nlsYFP-RXR remains exclusively in the cytoplasm. Coexpression of nlsRXR-BFP with GFP-VDR caused cytoplasmic retention of unliganded GFP-VDR, but calcitriol addition induced nuclear accumulation of both. These findings suggest that RXR dominates the nuclear import of unliganded VDR and this effect may be important for the ligand-independent functions of VDR, including the effect on hair growth. We and others have observed that ligand binding induces formation of multiple nuclear foci of GFP-GR, GFP-VDR, YFP-RXR, and GFP-ER, but the physiological importance of these foci remained to be elucidated. Studies on a cell line harboring a large array of MMTV-LTR allowed direct observation of human GR binding to this array. Mutational analysis demonstrated a correlation between hormone-dependent nuclear foci formation and DNA binding for both the VDR and the GR. In addition, fluorescence energy transfer experiments (FRET) revealed that calcitriol induces intranuclear foci formation of VDR/RXR heterodimers. Because VDR and RXR bind to DNA as heterodimers, this finding also suggests a correlation between focal receptor accumulation and DNA-binding. To study hormone-receptor interactions in living cells, we developed photostable and biologically active red fluorescing derivatives of vitamin D and used these labeled hormones together with fluorescent protein chimeras of VDR. Our studies revealed that calcitriol uptake into the cytoplasm is mediated, at least in part, by VLDL-type receptor-mediated endocytosis. This endocytosis is temperature sensitive, ATP-dependent, requires an acidic compartment, and is blocked by either receptor-associated protein or by the expression of the K44A mutant dynamin. Nuclear uptake of calcitriol depends on the presence of an intact VDR. Studies with import or export mutants of VDR indicated that shuttling VDR is essential for carrying the hormone into and out of the nucleus. Reverse FRET experiments supported this model. Our current efforts are directed towards characterizing receptor export mechanisms and the regulation of hormone targeting by chaperone proteins.