We use here a chimera of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to test the notion that the protein chaperone heat shock protein-90 (hsp90) is required for steroid-dependent translocation of the receptor through the cytoplasm along cytoskeletal tracks. The GFP-GR fusion protein undergoes steroid-mediated translocation from the cytoplasm to the nucleus, where it is transcriptionally active. Treatment of 3T3 cells containing steroid-bound GFP-GR with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, inhibits dexamethasone-dependent translocation from the cytoplasm to the nucleus. The t1/2 for translocation in the absence of geldanamycin is approximately 5 min, and the t1/2 in the presence of geldanamycin is approximately 45 min. In cells treated for 1 h with the cytoskeletal disrupting agents colcemid, cytochalasin D, and beta,beta'-iminodipropionitrile to completely disrupt the microtubule, microfilament, and intermediate filament networks, respectively, the GFP-GR still translocates rapidly to the nucleus in a strictly dexamethasone-dependent manner but translocation is no longer affected by geldanamycin. After withdrawal of the cytoskeletal disrupting agents for 3 h, normal cytoskeletal architecture is restored, and geldanamycin inhibition of dexamethasone-dependent GFP-GR translocation is restored. We suggest that in cells without an intact cytoskeletal system, the GFP-GR moves through the cytoplasm by diffusion. However, under physiological conditions in which the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery that is dependent upon hsp90 chaperone activity. In contrast to the GR, GFP-STAT5B, a signaling protein that is not complexed with hsp90, undergoes GH-dependent translocation to the nucleus in a manner that is not dependent upon hsp90 chaperone activity.