OBJECTIVE: In obese subjects, chronically elevated plasma concentrations of non-esterified fatty acids (NEFAs) exert a marked risk to contract insulin resistance and subsequently type 2 diabetes. When NEFA is acutely increased due to i.v. infusion of lipid, glucose disposal during a hyperinsulinemic-euglycemic clamp is reduced. This effect has been explained by a NEFA-induced decrease in skeletal muscle insulin sensitivity caused by accumulation of the lipid intermediates such as ceramide and diacylglycerol in the myocytes. However, neither the lipid-induced reduction of glucose disposal nor the intramyocellular lipid deposition has been compared directly in obese females and males. DESIGN: We studied eight obese females and eight obese males (body mass index (BMI): 32.6+/-1.4 and 32.8+/-0.8 respectively, non significant (NS)) matched for cardiorespiratory fitness relative to lean body mass (43.7+/-1.6 and 47.6+/-1.3 ml/kg min respectively, NS). METHODS: Each subject underwent two hyperinsulinemic-euglycemic clamps with infusion of lipid or saline respectively. Furthermore, the subjects exercised during the last half an hour of each clamp. RESULTS: The lipid-induced reduction in glucose disposal during the clamp was similar in females and males (46+/-10 and 60+/-4% respectively, NS). However, whole-body insulin sensitivity as well as non-oxidative glucose disposal was higher in obese females compared with obese males both during lipid and saline infusion (P<0.001 and P=0.01 respectively). Muscle ceramide, triacylglycerol (TAG), diacylglycerol (DAG), and glycogen content were similar between sexes and remained unchanged during the clamp and when exercise was superimposed. CONCLUSIONS: The lipid-induced inhibition of glucose disposal is similar in obese females and males. However, obese females are more insulin sensitive compared with obese males (both during saline and lipid infusion), which is not due to differences in the concentration of the muscle lipid intermediates such as ceramide and DAG.