This study investigated the effects of L-carnitine supplementation on muscle carnitine and glycogen content during submaximal exercise (EX). Triglycerides were evaluated by a fat feeding (90 g fat) and 3 h later subjects cycled for 60 min at 70% VO2max (CON). Muscle biopsies were obtained preexercise and after 30 and 60 min of EX. Blood samples were taken prior to and every 15 min of exercise. Subjects randomly completed two additional trials following 7 and 14 d of carnitine supplementation (6 g.d-1). During one of the two trials, subjects received 2000 units of heparin 15 min prior to EX to elevate FFA (CNhep); no heparin was administered during the other trial (CN). There were no differences in VO2, respiratory exchange ratio, heart rate, or g.min-1 of CHO and fat oxidized among the three trials. At rest serum total acid soluble (TASC) and free (FC) carnitine increased with supplementation (TASC; CON, 71.3 +/- 2.9; CN, 92.8 +/- 5.4; CNhep, 109.8 +/- 3.5 mumol.l-1) (FC; CON, 44.1 +/- 2.7; CN, 66.1 +/- 5.3; CNhep, 77.1 +/- 4.1 mumol.l-1). During EX, TASC remained stable, while FC decreased and short-chain acylcarnitine (SCAC) increased (P < 0.05). Muscle carnitine concentration at rest was unaffected by supplementation. During EX, muscle TASC did not change, FC decreased, and SCAC increased significantly in all three trials. Pre-EX and post-EX muscle glycogens were not different. Increased availability of serum carnitine does not result in an increase in muscle carnitine content nor does it alter lipid oxidation. It appears that there is an adequate amount of carnitine present within the mitochondria to support lipid oxidation.