The effects of cortisol on insulin sensitivity were examined in rats with the euglycaemic, hyperinsulinaemic clamp technique. Uptake of 2-deoxyglucose and incorporation of glucose into glycogen was followed in the white gastrocnemius, extensor digitorum longus, red gastrocnemius and soleus muscles as well as the liver (only glycogen synthesis). Maximal velocity and fractional velocity of the insulin-sensitive part of glycogen synthase (FV %) was measured in the muscles, as well as muscle fibre composition and capillary density. After 24 h exposure to cortisol, insulin sensitivity was diminished in the clamp measurements. This was paralleled by a decrease in glycogen synthesis in the most insulin-sensitive red gastrocnemius and Soleus muscles, but not in the white gastrocnemius or extensor digitorum longus muscles or the liver, and no effect was seen on 2-deoxyglucose uptake in muscles. FV % was markedly inhibited in all muscles. After 48 h exposure to cortisol, glycogen synthesis was markedly inhibited in all muscles, and 2-deoxyglucose uptake in all except the least insulin-sensitive muscle, WG. No changes in muscle morphology were found. These results suggest that the insulin resistance caused by cortisol is elicited in a stepwise manner, starting with an inhibition in the glycogen synthesis system in insulin-sensitive muscles, later including all muscles as well as 2-deoxyglucose uptake. This occurs without changes in morphology.