We have recently demonstrated that insulin stimulates muscle total carnitine (TC) accumulation during hypercarnitinaemia in humans, but hypercarnitinaemia per se had no effect (1). Given the essential role of carnitine in the integration of fat and carbohydrate metabolism, the aim of the present study was to investigate the effect of an increase in muscle TC content on skeletal muscle intermediary metabolism in resting humans. Seven healthy men (age 22.4 ± 1.5 y, BMI 26.1 ± 1.6 kg/m²) volunteered for the present study. On two randomised occasions, separated by 14 days, subjects underwent a 6 h euglycaemic hyperinsulinaemic clamp (2; 105 mU/m²/min), aimed at maintaining a physiologically high serum insulin concentration, which indicated the start of a 24 h experimental period. After 1 h, the insulin clamp was accompanied by iv infusion of L-carnitine (CARN; 15 mg/kg bolus followed by 10 mg/kg/h) or the equivalent volume of saline (CON) for 5 h. Thereafter, subjects were fed a standardised, carnitine free meal (approx. 1500 kcal; 55% carbohydrate, 35% fat, and 10% protein). Arterialised-venous blood samples were obtained every hour during each clamp (and the following morning after an overnight fast), and needle biopsy samples were obtained from the vastus lateralis muscle immediately before and after each clamp and the following morning. Statistical analysis was performed using repeated-measures two-way ANOVA and paired Student’s t tests. Data are expressed as means ± SEM. The hyperinsulinaemic clamp produced similar steady state serum insulin concentrations of 160.1 ± 1.9 and 155.8 ± 3.9 mU/l during the CON and CARN infusion visits, respectively. The combination of steady-state hypercarnitinaemia (633.5 ± 10.6 μmol/l) and hyperinsulinaemia increased muscle TC content from 22.5 ± 2.0 to 26.6 ± 1.6 mmol/(kg dm) (P<0.01), and was associated with a decrease in pyruvate dehydrogenase complex activity (1.1 ± 0.1 vs. 0.7 ± 0.1 mmol acetyl-CoA/min/(kg wm); P<0.05) and muscle lactate content (10.4 ± 2.5 vs. 6.0 ± 1.0 mmol/(kg dm); P<0.05), and an overnight increase in muscle glycogen (567 ± 22 vs. 736 ± 24 mmol/(kg dm); P<0.01) and long-chain acyl-CoA content (12.1 ± 3.0 vs. 19.8 ± 3.5 mmol/(kg dm); P<0.05) compared to CON. Muscle TC content was 25.1 ± 2.2 mmol/(kg dm) following the overnight fast. This study suggests that an acute increase in skeletal muscle TC content in humans results in an inhibition of carbohydrate oxidation in conditions of high carbohydrate availability, which is probably due to a carnitine-mediated increase in fat oxidation. These novel findings may be of importance to the regulation of muscle fat oxidation, particularly during exercise when carnitine availability may limit fat oxidation and in obesity and type 2 diabetes where it is known to be impaired.