Carnitine is known to accumulate in brain, therefore transport of carnitine through the blood-brain barrier was studied in an in vitro system using bovine brain capillary endothelial cells (BBCEC) grown on filter inserts in a co-culture system with glial cells. Long-term exposure of BBCEC to carnitine resulted in a high accumulation of long-chain acyl carnitines, which decreased dramatically upon removal of carnitine. Kinetic analysis of carnitine accumulation indicated a possibility of functioning of more than one transporter. BBCEC were incubated in the presence of substrates and inhibitors of known carnitine transporters added from either apical or basolateral side. Inhibition by replacement of sodium and expression of OCTN2 (RT-PCR) were in agreement with earlier reports on the functioning of OCTN2 in apical membrane. For the first time, functioning of OCTN2 was demonstrated in the basolateral membrane, as well as functioning in both membranes of a low affinity carnitine transporter B 0,+ . Expression of B 0,+ in BBCEC was confirmed by RT-PCR. These results suggest that OCTN2 and B 0,+ could be involved in carnitine transport in both the apical and basolateral membrane.