Exercise stimulates muscle glucose uptake both directly and by increasing the sensitivity of this process to insulin. This study was designed to investigate whether the level of dietary fat would interact with the action of acute exercise in the presence or absence of insulin. Weanling female Sprague-Dawley rats were fed two levels of dietary fat (5 vs. 20%; wt/wt) for 6 wk. Rats then remained sedentary or were exercised by a single bout of swimming for a total of 2 h with 5-min rest intervals each 0.5 h. Basal (insulin-independent) and insulin-stimulated glucose uptake rates were determined in isolated epitrochlearis muscles by using 3-O-[methyl-3H]methyl-D-glucose. Muscles of sedentary rats fed a high-fat diet showed decreased glucose uptake overall because of a marked decrease in insulin-stimulated uptake. Muscles of rats fed a high-fat diet also showed considerable impairment of insulin-dependent glucose uptake measured both immediately and 3.5 h after exercise. Glucose uptake was suppressed by 64% at half-maximal concentrations of insulin (0.8 nM) and by 34% at maximally stimulating (20 nM) insulin levels. This lowered sensitivity and response to insulin were not altered by acute exercise. Glucose uptake in response to acute exercise (insulin independent) was quantitatively similar in rats fed high- and low-fat diets; rats on high-fat diet, however, showed higher insulin-independent glucose uptake at 3.5 h after exercise.