This study was undertaken to challenge the hypothesis that short-term administration of carnitine during exercise can modify skeletal muscle carnitine homeostasis and fuel metabolism in normal humans. With a randomized, blinded, crossover design, subjects received carnitine or placebo at the start of a bicycle ergometer exercise session. During the 2 hours after intravenous administration of 185 mumol/kg carnitine, carnitine kinetics could be described with a central compartment volume of distribution of 200 ml/kg, a total clearance from this compartment of 1.9 ml/min/kg, and a renal clearance of 1.3 ml/min/kg. Carnitine administration had no effect on muscle total carnitine content or the workload-dependent accumulation of acylcarnitines in skeletal muscle. Carnitine had no effect on the respiratory exchange ratio, muscle lactate accumulation, plasma lactate concentration, muscle glycogen utilization, or plasma beta-hydroxybutyrate concentration during exercise. Thus the skeletal muscle carnitine pool is segregated from dramatic changes in the plasma carnitine pool, and short-term administration of carnitine has no significant effect on fuel metabolism during exercise in humans.