Ethanol consumption has been associated with glucose intolerance and insulin resistance and is suggested to be an independent risk factor in the development of non-insulin-dependent diabetes mellitus. We have investigated the long-term effects of ethanol consumption on insulin-regulated glucose transport in rat adipocytes. Male Wistar rats were fed a high-fat liquid diet containing 35% ethanol (ethanol fed) or a control diet that isocalorically substituted maltose dextrin for ethanol (ad libitum). A third group was pair fed the control diet. Basal rates of 2-deoxyglucose uptake were similar in adipocytes from all three groups. Treatment with insulin increased 2-deoxyglucose uptake in ad libitum- and pair-fed rats but did not stimulate uptake in ethanol-fed rats. Similarly, although okadaic acid increased 2-deoxyglucose uptake in pair-fed rats, it had no effect in ethanol-fed rats. GLUT-1 quantity was greater in pair-fed and ethanol-fed rats compared with ad libitum controls. GLUT-4 was decreased in ethanol-fed compared with pair-fed rats but was not different from ad libitum controls. In ad libitum- and pair-fed rats, insulin increased the translocation of GLUT-4 to the cell surface by 2.0-fold. In contrast, translocation of GLUT-4 was not observed after insulin stimulation of ethanol-fed rats, paralleling the loss of insulin-stimulated glucose uptake. In ethanol-fed rats, GLUT-4 protein quantity was negatively associated with increased Gs alpha protein and isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate production. These data suggest that loss of insulin-stimulated glucose uptake in rat adipocytes after chronic ethanol feeding is at least partially due to decreased movement of GLUT-4 to the cell surface after insulin stimulation.