We have previously shown in primary cultured adipocytes that chronic insulin exposure decreases insulin’s sub-sequent ability to maximally restimulate the glucose transport system, and that extracellular glucose potentiates this ligand- induced defect in maximal insulin responsiveness. To examine whether glucose could also modulate insulin sensitivity (i.e. acute insulin effects at submaximal concentrations), adipocytes were cultured for 5 and 24 h in the absence and presence of various glucose and insulin concentrations. Then, after washing cells to remove any insulin and allow for full deactivation of transport, we assessed the dose response of insulin’s acute ability to stimulate 2-deoxyglucose transport, bind to cell surface receptors, and activate insulin receptor tyrosine kinase activity. After 5 h, glucose and insulin alone had no chronic regulatory effects; however, in combination, these agents were able to decrease insulin sensitivity. In cells preincubated with 50 ng/ml insulin, the insulin ED50for acute stimulation of glucose transport was increased by 65% and 116% as medium glucose was raised to 5 and 20 mM, respectively, relative to that at 0 mM glucose. After 24 h, chronic exposure to either glucose (20 mM) or insulin (50 ng/ml) alone increased the ED50value by 52%, and, together they acted synergistically to increase the ED50by 183%. While glucose and insulin independently and synergistically impaired insulin sensitivity, both agents were necessary for coregulation of maximal insulin responsiveness (confirming our previous observation). Insulin receptor down-regulation (18%) was observed after 24 h (but not 5 h) in insulin-treated cells; however, the major portion of the decrease in insulin sensitivity was due to uncoupling of occupied insulin receptors from stimulation of the glucose transport system. To further determine the mechanism for postbinding desensitization, we tested for concordant regulation of insulin receptor kinase activity. Insulin’s ability to stimulate the receptor tyrosine kinase was assessed by multiple methods, including 1) receptor autophosphorylation and phosphorylation of Glu4-Tyr1by solubilized insulin receptors acti-vated in vitro, 2) histone-2B phosphorylation by receptors that were stimulated in intact cells and then solubilized under conditions that preserve the in cellulo phosphorylation state, and 3) receptor autophosphorylation and phosphorylation of ppl80 in intact cells. Long term treatment (24 h) with glucose (10 mM) and insulin (50 ng/ml) markedly decreased insulin sensitivity (and receptor coupling), but did not affect insulin receptor kinase activity in any of these studies. We conclude that 1) glucose and insulin can independently and synergistically regulate insulin sensitivity by uncoupling ligand-bound insulin receptors from stimulation of glucose transport, and 2) glucose/insulin-induced desensitization occurred without any effect on insulin receptor tyrosine kinase activity. These observations suggest that decreased insulin sensitivity in target tissue may be induced by hyperglycemia and hyperinsulinemia in noninsulin-dependent diabetes mellitus via a receptor kinase-independent mechanism. © 1991 by The Endocrine Society.