L-carnitine (LC) resides mainly in skeletal muscle and its Na⁺ dependent transport occurs via the organic cation transporter OCTN2 (Tamai et al. 1998). Investigation of the effect of increasing muscle LC content on energy metabolism is warranted based on evidence that free LC availability may limit muscle fat oxidation during incremental exercise (van Loon et al. 2001). A limited amount of research has however addressed whether an increase in human muscle LC content is achieved by LC administration. This study aimed to determine whether insulin, which is known to increase Na⁺/K⁺ pump activity (Clausen, 2003), could augment LC accumulation in human muscle. Eight healthy men (age 22.4 ± 0.4 years, B.M.I. 22.8 ± 0.5 kg m^-2) volunteered for the present study, which was approved by the local ethics committee. On two randomised occasions, separated by 14 days, subjects underwent a 6 h euglycaemic insulin clamp (5 and 105 mU m^-2 min^-1) aimed at maintaining fasting or physiologically high plasma insulin concentrations. After 1 h, each insulin clamp was accompanied by intravenous infusion of LC (15 mg kg^-1 bolus followed by 10 mg kg^-1 h^-1) for 5 h. Arterialised-venous blood samples were obtained every hour and needle biopsy samples were obtained from the vastus lateralis immediately before and after each insulin clamp. Statistical analysis was performed using repeated measures one- and two-way ANOVA. Data are expressed as means ± S.E.M. The 5 and 105 mU m^-2 min^-1 insulin clamp produced a steady state serum insulin concentration of 7.1 ± 0.4 and 149.2 ± 6.9 mU l^-1, respectively. Plasma LC concentration remained above 450 μmol l^-1 throughout LC infusion, but was lower after 4 and 5 h of infusion during the 105 mU m^-2 min^-1 insulin clamp (P<0.01). Muscle total LC did not change when insulin was maintained at a fasting concentration (pre 22.4 ± 1.0 vs post 22.7 ± 1.1 mmol kg^-1 dry weight), but increased from 22.0 ± 0.9 to 24.6 ± 1.4 mmol kg^-1 during the 105 mU m^-2 min^-1 insulin clamp (P<0.05), which was associated with a 2.3 ± 0.3-fold increase in OCTN2 mRNA expression (n = 5; P<0.01). The present findings demonstrate that maintaining a supra-physiological plasma LC concentration for 5 hrs has no effect on muscle total LC concentration or OCTN2 mRNA expression. However, increasing plasma LC availability in the presence of elevated serum insulin increases muscle LC concentration and OCTN2 transcription. We believe this is a result of insulin increasing sarcolemmal Na⁺/K⁺ pump activity and thereby Na⁺-coupled transport of LC via OCTN2.