More than 95% of the body’s total carnitine store resides within skeletal muscle, where it plays two essential roles (for review see Stephens et al. 2007). Firstly, during intense exercise, when the rate of pyruvate dehydrogenase complex (PDC) flux exceeds the rate of acetyl group utilisation by the tricarboxcylic acid cycle, free carnitine buffers acetyl group production to maintain the mitochondrial acetyl-CoA/CoASH ratio. Secondly, free carnitine is essential for the translocation of long-chain fatty-acids into the mitochondrial matrix for subsequent beta-oxidation. This presentation will highlight research demonstrating the significance of muscle carnitine availability to the regulation of muscle fuel metabolism in vivo at rest in normal and insulin resistant states. It will also draw attention to evidence published over the past decade that high muscle PDC flux and thereby acetylcarnitine accumulation during exercise can reduce muscle free carnitine availability to an extent that it will limit mitochondrial fat oxidation in vivo (van Loon et al. 2001). Oral L-carnitine feeding has been advocated as an ergogenic aid, however L-carnitine feeding per se has no impact on muscle total carnitine content, fuel metabolism or exercise performance. We have demonstrated that intra-venous L-carnitine infusion (steady-state plasma [550-600] µmol.l-1) under insulin clamp conditions acutely increased muscle total carnitine (by ~15%) in health male volunteers when serum insulin concentration was increased above 50 mU.l-1. Furthermore, this reduced muscle glycolysis, blunted PDC activation (under conditions of fixed glucose delivery), increased glycogen storage, and increased muscle long-chain acyl-CoA content at rest (for review see Stephens et al. 2007). More recently, we have shown this increase in muscle total carnitine content can be achieved by combined carbohydrate and L-carnitine feeding over a 6 month period in healthy, male volunteers. Moreover, this increase in TC content reduced PDC activation and muscle glycogen utilisation during low intensity exercise (50% VO2max), and maintained PDC flux and improved exercise performance during more intense exercise (80% VO2max; Wall et al. 2011). Conversely, we have recently found that muscle total carnitine content can be markedly depleted by blocking renal carnitine retention using mildronate, which had the effect of reducing whole body fat oxidation and increasing muscle glycogen utilisation in a rodent model of obesity (Porter et al. unpublished observation). Collectively these findings point to a central role for carnitine in the regulation of muscle fuel selection at rest and during exercise in obese and non-obese states. The demonstration that muscle carnitine availability can be readily manipulated in humans, and significantly impacts on physiological function under a variety of conditions, will result in renewed interest in this compound.