Activation of the sympathetic nervous system inhibits insulin-stimulated glucose uptake. However, the underlying mechanisms are incompletely understood. Therefore, we studied the effects of catecholamines on insulin-stimulated glucose uptake and insulin-stimulated translocation of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes. We found that epinephrine (1 µM) nearly halved insulin-stimulated 2-deoxyglucose uptake. The -adrenoceptor antagonist propranolol (0.3 µM) completely antagonized the inhibitory effect of epinephrine on insulin-stimulated glucose uptake, whereas the -adrenoceptor antagonist phentolamine (10 µM) had no effect. When norepinephrine was used instead of epinephrine, the results were identical. None of the individual selective -adrenoceptor antagonists (1 µM, ₁: metoprolol, ₂: ICI-118,551, ₃: SR 59230A) could counteract the inhibitory effect of epinephrine. Combination of ICI-118,551 and SR 59230A, as well as combination of all three selective -adrenoceptor antagonists, abolished the effect of epinephrine on insulin-stimulated glucose uptake. Following differential centrifugation we measured the amount of GLUT-1 and GLUT-4 in the plasma membrane and in intracellular vesicles by means of Western blotting. Both epinephrine and norepinephrine reduced the insulin-stimulated GLUT-4 translocation to the plasma membrane. These results show that -adrenergic (but not -adrenergic) stimulation inhibits insulin-induced glucose uptake in 3T3-L1 adipocytes most likely via the ₂- and ₃-adrenoceptor by interfering with GLUT-4 translocation from intracellular vesicles to the plasma membrane.