Juvenile visceral steatosis (JVS) mice, which show systemic l-carnitine deficiency, may be an animal model of Reye's syndrome because of its phenotype of fat deposition and mitochondrial abnormalities in the liver. In this study, we compared the characteristics of the l-carnitine transport in isolated hepatocytes from wild-type and JVS mice. The uptake of l-carnitine by wild-type hepatocytes was saturable and the Eadie-Hofstee plot showed 2 distinct components. The apparent Michaelis constant (Km) and the maximum transport rate (Vmax) were 4.6 μmol/L and 59.5 pmol/15 min/106 cells, respectively, for the high-affinity component, and 404 μmol/L and 713 pmol/15 min/106 cells, respectively, for the low-affinity component. The high-affinity l-carnitine uptake occurred via an active carrier-mediated transport mechanism, which is characterized by Na+-, energy-, and pH-dependency. On the other hand, the high-affinity uptake was absent in JVS hepatocytes, and the values ofKm andVmaxfor the low-affinity uptake were 475 μmol/L and 557 pmol/15 min/106 cells, respectively. The hepatic carnitine transport properties in wild-type hepatocytes were similar to those of high-affinity mouse Octn2-transfected HEK293 cells. This study suggests that Octn2-type carnitine transporter is dysfunctional in hepatocytes of JVS mice.