Background/Aims: Juvenile visceral steatosis (jvs–/–) mice lack the activity of the carnitine transporter OCTN2 and are dependent on carnitine substitution. The effects of carnitine deprivation on carnitine homeostasis and energy metabolism are not known in jvs–/– mice. Methods: jvs–/– mice were studied 3, 6 and 10 days after carnitine deprivation, and compared to jvs–/– mice substituted with carnitine, wild-type (jvs+/+) and jvs+/– mice. Carnitine concentrations were assessed radioenzymatically. Results: Compared to wild-type mice, carnitine-treated jvs–/– mice had decreased plasma β-hydroxybutyrate levels and showed hepatic fat accumulation. The carnitine levels in plasma, liver and skeletal muscle were decreased by 58, 16 and 17%, respectively. After ten days of carnitine deprivation, the plasma carnitine concentration had fallen by 87% (to 2.3 µmol/l) and the tissue carnitine levels by ≈50% compared to carnitine-treated jvs–/– mice. Carnitine deprivation was associated with a further drop in plasma β-hydroxybutyrate and increased hepatic fat. Skeletal muscle glycogen stores decreased and lactate levels increased with carnitine deprivation, whereas tissue ATP levels were maintained. Conclusions: In jvs–/– mice, tissue carnitine stores are more resistant than carnitine plasma concentrations to carnitine deprivation. Metabolic changes (liver steatosis and loss of muscle glycogen stores) appear also early after carnitine deprivation.