Plasma membrane vesicles prepared from rat brain contain a saturable, high-affinity transport system for L-glutamine that exhibits the following characteristics: (1) The rate of L-glutamine transport is linear up to 200 μ/mL membrane protein. (2) Transport of [3H]-L-glutamine is linear with time for at least 10 min, is significantly reduced by lowering the assay temperature to 4 °C, and is essentially abolished by the addition of excess unlabeled L-glutamine. (3) The transport rate is optimal in the range of pH 7.4–8.2. (4) The system exhibits a Km for L-glutamine of ~ 1.7 μM and a Vmax of ~46 pmol/(min·mg of protein). (5) The system is not highly dependent upon the addition of monovalent or divalent cations. (6) Inhibitor studies reveal that the amino acid amides exhibit the highest affinity for the system and that there is a high specificity for the L-isomers.