To test the hypothesis that glucocorticoids reduce insulin-stimulated skeletal muscle glucose transport by inhibiting the recruitment of GLUT4 glucose transporters to the cell surface, we determined the effect of glucocorticoid treatment on cell-surface GLUT4 using the impermeant glucose transporter photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-[2-³ H]1,3-bis-(d-mannos-4-yloxy)-2-propylamine (ATB-[2-³H]BMPA), and GLUT4 immunoprecipitation. Male Sprague-Dawley rats were treated with dexamethasone ([Dex] 0.9 mg/kg for 2 days) and compared against pair-fed controls. 2-[³H]deoxyglucose (2-[³H]DG) uptake in isolated soleus muscles was measured under conditions in which uptake reflects glucose transport activity. In control muscles, 2-[³H]DG uptake was stimulated eightfold by insulin (20 nmol/L). Dex treatement reduced maximal insulin-stimulated 2-[³H]DG uptake by 48% ± 4% (mean ± SEM) and decreased cell-surface (ATB-[2-³H]BMPA—photolabeled) GLUT4 by 48% ± 3%, despite an increase in total muscle GLUT4 content of 26% ± 7%. These findings indicate that glucocorticoid-induced inhibition of insulin-stimulated glucose transport in muscle is due to impaired recruitment of GLUT4 to the cell surface.