Previous results from our laboratory demonstrated that chronic administration of fructose to normal rats led to both hyperinsulinemia and in vivo insulin resistance. To localize the major tissue site of insulin resistance in fructose-fed animals, we compared glucose uptake by perfused hindlimb skeletal muscle and liver from rats fed either a 60% fructose diet or laboratory chow. Glucose uptake by perfused muscle from chow and fructose-fed rats was comparable at perfusate insulin levels of 0 microunit/ml (15.2 versus 15.5 microliters/min/g muscle), 100 microunits/ml (18.3 versus 19.8), and greater than 500 microunits/ml (35.5 versus 33.4). In contrast, glucose outflow from livers of fructose-fed rats was significantly greater (p less than .02) than chow-fed animals perfused in the absence of added insulin (52.1 versus 36.5 mumol/g). Furthermore, the ability of insulin to suppress glucose outflow was less in livers from fructose-fed rats at perfusate insulin level of 165 microunits/ml (13.2 versus 41.4% as well as at insulin concentration greater than 900 microunits/ml, (32.5% versus 62.2%). These findings suggest that the insulin resistance resulting from chronic fructose feeding is due to the diminished ability of insulin to suppress hepatic glucose output, and not to a decrease in insulin-stimulated glucose uptake by muscle.