To test the hypothesis that glucocorticoids inhibit muscle glucose transport apart from changes in early insulin-signaling events, we determined the effect of glucocorticoid treatment on the activation of glucose transport by both insulin and non-insulin-related stimuli (insulin-like growth factor [IGF] I and hypoxia) in rat skeletal muscle. Male Sprague-Dawley rats were treated with dexamethasone (Dex) (0.8 mg/kg for 2 days) and compared with 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 10- fold by insulin (10 nmol/l) or IGF-I (50 nmol/l) and sixfold by hypoxin. Dex treatment decreased 2-[3H]DG uptake at all concentrations of insulin tested, reducing maximal insulin-stimulated 2-[3H]DG uptake by 41 ± 11% (mean ± SE, P < 0.05) and basal 2-[3H]DG uptake by 38 ± 6% (P < 0.01). Dex treatment also inhibited 2-[3H]DG uptake at all concentrations of IGF-I tested, reducing maximal IGF-I-stimulated 2-[3H]DG uptake by 29 ±+ 2% (P < 0.01), and decreased hypoxia-stimulated 2-[3H]DG uptake by 61% (P < 0.01). Dex treatment increased soleus GLUT4 protein content by 11%. Thus, Dex treatment reduces basal glucose transport and decreases the maximal response of skeletal muscle glucose transport to insulin, the related hormone IGF-I, and the non-insulin-related stimulus hypoxia. These findings support the hypothesis that, in addition to altering early insulin-signaling events, glucocorticoids may also act by inhibiting the glucose transport system, per se, perhaps by affecting GLUT4 subcellular trafficking.