To study the effect of carnitine depletion on physical working capacity, healthy subjects were administered pivaloyl-conjugated antibiotics for 54 days. The mean carnitine concentration in serum decreased from 35.0 to 3.5 mmicromol/L, and in muscle from 10 to 4.3 micromol/g noncollagen protein (NCP). Exercise tests were performed before and after 54 days' administration of the drug. At submaximal exercise, there was a slight increase in the concentration of 3-hydroxybutyrate in serum, presumably caused by decreased fatty acid oxidation in the liver. There was also a decreased consumption of muscle glycogen, indicating decreased glycolysis in the skeletal muscle. The muscle presumably had enough energy available, since there was no significant decrease in the concentration of adenosine triphosphate (ATP) and creatine phosphate during exercise. The work at maximal oxygen uptake (VO2max) and the maximal heart rate were reduced. Since VO2max is considered dependent on heart function, carnitine depletion seemed to affect cardiac function.