Exercise can induce short-term increases in the sensitivity and responsiveness of skeletal muscle glucose transport to insulin. The purpose of this study was to determine the effect of carbohydrate deprivation on the persistence of increased insulin sensitivity and responsiveness after a bout of exercise. Three hours after a bout of exercise, epitrochlearis muscles from carbohydrate-deprived (fat fed) rats showed a 25% greater increase in 3-O-methylglucose (3-MG) transport in response to a maximal insulin stimulus compared with muscles of nonexercised rats; this increase in insulin responsiveness had reversed 18 h postexercise. Muscles of rats fed carbohydrate showed no increase in insulin responsiveness 3 h after exercise. The effect of 60 microU/ml of insulin on 3-MG transport was approximately twofold greater in muscles studied 3 h after exercise than in nonexercised controls regardless of dietary carbohydrate intake. This increase in insulin sensitivity was lost within 18 h in carbohydrate-fed rats but persisted for at least 48 h in carbohydrate-deprived rats. Muscle glycogen increased approximately 41 mumol/g in the rats fed carbohydrate for 18 h, and only approximately 14.5 mumol/g in the rats fed fat for 48 h, after exercise. The persistent increase in insulin sensitivity after exercise in carbohydrate-deprived rats was unrelated to caloric intake, as muscles of fasted and fat-fed rats behaved similarly.