The association between nuclear distribution and mobility of the human glucocorticoid receptor was examined in living COS-1 cells using yellow fluorescent protein- and cyan fluorescent protein-tagged receptors. Quantitation of the nuclear distribution induced by an array of glucocorticoid ligands revealed a continuum from a random (cortisone) to a nonrandom (triamcinolone acetonide) receptor distribution. Structure-function analysis revealed that the 9-fluoro and 17-hydroxy groups on the steroid significantly impact nuclear receptor distribution. Using time-lapse microscopy, the triamcinolone acetonide-induced receptor distribution did not change significantly over a period of 15 sec. However, using fluorescence recovery after photobleaching, the individual receptors moved at a much faster rate, indicating rapid exchange of receptors on immobile nuclear subdomains. Receptor mobilities for 13 different steroids, measured by fluorescence recovery after photobleaching, appeared to correlate with receptor distribution. Ligands that induced a nonrandom distribution induced slower receptor mobility and vice versa. Finally, application of 2-photon confocal microscopy revealed differences in receptor mobility between nuclear subdomains. Areas of high receptor concentration showed slower mobility than areas of low receptor concentration. Thus, glucocorticoid receptors can be targeted (depending on the ligand) to relatively immobile nuclear subdomains. The transient association of receptor with these domains decreases the mobility of the receptor.