The system L-amino acid transporter is a major nutrient transport system that is responsible for Na+-independent transport of neutral amino acids including several essential amino acids. We have compared and examined the expressions and functions of the system L-amino acid transporters in both rat astrocyte cultures and C6 glioma cells. The rat astrocyte cultures expressed the l-type amino acid transporter 2 (LAT2) with its subunit 4F2hc, whereas the l-type amino acid transporter 1 (LAT1) was not expressed in these cells. The C6 glioma cells expressed LAT1 but not LAT2 with 4F2hc. The [14C]l-leucine uptakes by the rat astrocyte cultures and C6 glioma cells were Na+-independent and were completely inhibited by the system l selective inhibitor, BCH. These results suggest that the transport of neutral amino acids including several essential amino acids into rat astrocyte cultures and C6 glioma cells are for the most part mediated by LAT2 and LAT1, respectively. Therefore, the rat astrocyte cultures and C6 glioma cells are excellent tools for examining the properties of LAT2 and LAT1, respectively. Moreover, the specific inhibition of LAT1 in cancer cells might be a new rationale for anti-cancer therapy.