To investigate the intracellular trafficking of glucocorticoid receptor (GR) in response to various conditions in a single living cell, a green fluorescent protein (GFP) and rat GR chimera construct (GFP-GR) was prepared. We transiently transfected GFP-GR into primary cultured rat hippocampal neurons, cortical glial cells, and non-neural cells, e.g. COS-1 cells and CV-1 cells, and compared the dynamic changes in subcellular localization of GFP-GR in these cells. When GFP-GR was expressed in the cells, GFP-GR efficiently transactivated the mouse mammary tumour virus promoter in response to dexamethasone (DEX). The cytoplasm-to-nuclear translocation of GFP-GR induced with 10–7 m DEX, a specific agonist of GR, at 37 °C was completed within 30 min in all cell types used, and the rate of nuclear translocation was dependent on the ligand dose. The translocation of GFP-GR into the nucleus from the cytoplasm was induced in a ligand-specific manner, similar to that of the native GR. The disruption of microtubules by colchicine or nocodazole showed no significant effect on the DEX-induced GFP-GR translocation from the cytoplasmic region to the nuclear region. The cells were not deteriorated during time-lapse imaging analysis for 1 h at 37 °C. The present findings suggest that the subcellular localization of GFP-GR is dynamically changed in response to extracellular and intracellular conditions, and that there are no conspicuous variations in the manner of trafficking of GR among different types of cells in vitro.