Our hypothesis was that insulin resistance for carbohydrate metabolism develops after a single day of muscular disuse. The immobilization of the mouse hindlimb for 24 h was used to produce muscular disuse (group c). As food intake was voluntarily decreased during the immobilization, two additional groups were used: group A was untreated and ate ad libitum, whereas group B was anesthetized with group C and was fed amounts of food similar to those eaten by group C. Because groups B and C differed only by limb immobilization, group B was used as the reference group. When insulin was present in the incubation media, the rates of 2-deoxyglucose uptake and glycogen synthesis were always significantly decreased in soleus muscles from group C (anesthetized, ate less, immobilized) as compared to group B (anesthetized, pair-fed food that group C ate). Significant interaction between the factors of insulin and muscular disuse for the rates of 2-deoxyglucose uptake and glycogen synthesis support the concept that disuse of skeletal muscle attenuates insulin action. These observations are a direct demonstration of a decrease in insulin responsiveness in skeletal muscle as the result of a single day of muscular inactivity. Because plasma insulin concentrations were significantly lower in groups B and C than in the untreated group, it seems likely that the development of insulin resistance in disused skeletal muscle is independent of plasma insulin levels.