Glucocorticosteroids are the most effective therapy for the suppression of airway inflammation. Classically, ligand binding to the inactive cytosolic receptor (GR) causes a conformational change enabling nuclear translocation of the active GR and alteration in the transcriptional response. We have investigated GR-transcription factor interactions within the cytoplasm where many transcription factors are localized prior to activation. Active DNA binding GR complexes were found within the cytoplasm of human lung epithelial cells (A549). Stimulation by dexamethasone (1 microM) caused translocation of active GR into the nucleus and inhibition of RANTES release. Coincubation of cells with dexamethasone and TNF alpha (1 ng/ml) or PMA (0.1 microM) prevented RANTES release and the translocation of activated GR to the nucleus without affecting GR levels as detected by western blotting. These results suggest that a major site of pro-inflammatory cytokine action may be within the cytoplasm of steroid-responsive cells acting via prevention of the GR translocation into the nucleus.