Since corticosteroid treatment is often accompanied by insulin resistance, we explored the role of corticosteroids in the regulation of the insulin effectiveness in cultured 3T3 (mouse) adipocytes. Exposure of the fat cells to dexamethasone or corticosterone (0–5 days) induced a time-, concentration-, and protein synthesis-dependent and reversible decrease in insulin binding and in basal and insulin-stimulated 2-deoxyglucose uptake. The decrease in binding (50%) was primarily due to a decrease in receptor affinity i. e. to an increase in the rate of dissociation of insulin from its receptors, and was independent from the effects of pH and temperature on the affinity. The reduction in the 2-deoxyglucose uptake (30–50%) was due to a decrease in the hexose transport capacity rather than to a decrease in the phosphorylation component of the 2-deoxyglucose uptake process. Lineweaver-Burk analysis revealed the dexamethasone induced a decrease in the apparent V_max of the transport system i. e. in the number or activity of the hexose transporters. The effect of dexamethasone seemed to be superimposed on that of long-term insulin treatment, suggesting a different mechanism. It is concluded that corticosteroids act as long-term regulators of the insulin effectiveness by influencing the rate at which insulin dissociates from its receptors and by altering the number or activity of the hexose transporters by a common mechanism, which differs from that of the long-term regulatory effect of insulin.