Catecholamines are known to have short-term regulatory effects on fat cell hexose uptake. We examined the long-term effects of catecholamines on the insulin-sensitive 2-deoxyglucose (dGlc) uptake in cultured 3T3-L1 adipocytes. Prolonged exposure (48 h) to isoproterenol (beta-adrenergic agonist) stimulated the basal dGlc uptake up to 90%. The effect was specific, time, concentration, and protein synthesis dependent and reversible. The effect of insulin was unaltered and superimposed on the increase in basal dGlc uptake. The long-term effect of isoproterenol was mimicked by epinephrine, dibutyryl cAMP (DBcAMP), and 1-methyl-3-isobutylxanthine (IBMX). By contrast, short-term exposure to isoproterenol (and epinephrine) induced a protein synthesis-independent increase in basal dGlc uptake (30%) not accompanied by an increase in insulin responsiveness. Moreover, on short-term basis, DBcAMP and IBMX suppressed both the basal and insulin-stimulated uptake up to 50%. Determination of the intracellular nonphosphorylated dGlc during the uptake and of the hexokinase activity revealed that the long-term effect of isoproterenol was most likely due to alterations low in dGlc transport. In conclusion, long-term regulators of hexose uptake are in cultured 3T3-L1 adipocytes, isoproterenol, and other cAMP stimulators. The long-term effect is independent from the short-term regulatory effect of the agents and from the effect of insulin.