We have studied the ability of dexamethasone to regulate the glucose transport system in primary cultured adipocytes and delineated the mechanisms of insulin resistance after both acute and chronic treatment. Acutely, 20 nM dexamethasone led to a 65% decrease in basal and a 31% decrement in maximally insulin-stimulated glucose transport (ED50 = 3-4 nM; t1/2 = 50 min). These effects were maximal by 90-120 min, and a plateau was maintained over an additional 1-1.5 h. Chronic dexamethasone exposure (24 h) led to a more profound decrease in basal (77%; ED50 = 0.4 nM) and maximally stimulated (55%; ED50 = 1.0 nM) rates of glucose transport and shifted the transport: Insulin dose-response curve to the right by increasing the half-maximally effective insulin concentration from 0.2 to 0.4 ng/ml. Dexamethasone did not affect cell surface insulin binding over 24 h. Both the short and long term effects of dexamethasone were partially blocked by the combined presence of insulin during preincubation and were not modulated by glucose. We also assessed effects on the number and cellular distribution of glucose transporter proteins using the cytochalasin- B binding assay. After 2 h, dexamethasone (30 nM) decreased the number of glucose transporters in plasma membranes by 30% in basal cells and by 41% in maximally insulinstimulated cells, while increasing the number of low density microsomal transporters by 22-23% (P = NS). Transporter number in a total cellular membrane fraction was unaltered by short term dexamethasone. Chronic dexamethasone exposure (24 h) decreased plasma membrane and low density microsomal transporters by 30-50% in both basal and insulin-stimulated cells and depleted transporters by 43% in a total cellular membrane fraction. In conclusion, 1) dexamethasone induces progressive insulin resistance by sequentially regulating multiple aspects of the insulin-responsive glucose transport system. At early times (2 h) dexamethasone impairs insulin's ability to translocate intracellular glucose transporters to the cell surface and with more chronic exposure (24 h), depletes the total number of cellular transporters. 2) Glucose modulates desensitization of the glucose transport system by insulin, but not by dexamethasone, and thus, there are both glucose-dependent and -independent mechanisms of insulin resistance. 3) Insulin can heterologously inhibit dexamethasone's effects on glucose transport at both early and late phases of desensitization. These studies highlight the complex hormonal regulation at the glucose transport system. © 1989 by The Endocrine Society.