Transport of l‐[³H]carnitine and acetyl‐l‐[³H]carnitine at the blood–brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl‐l‐[³H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl‐l‐carnitine and in the absence of sodium ions. Similar transport properties for l‐[³H]carnitine and/or acetyl‐l‐[³H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of l‐[³H]carnitine and acetyl‐l‐[³H]carnitine by RBEC1 were sodium ion‐dependent, saturable with K_m values of 33.1 ± 11.4 µm and 31.3 ± 11.6 µm, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT‐PCR method. Furthermore, the uptake of acetyl‐l‐[³H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of l‐[³H]carnitine and acetyl‐l‐[³H]carnitine in jvs mice were slightly lower than those of wild‐type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of l‐carnitine and acetyl‐l‐carnitine from the circulating blood to the brain across the BBB.