Uptakes of neutral L-amino acids into rabbit choroid plexus apical membrane vesicles were studied using L-[14C]amino acids. Apical membrane vesicles were prepared using Ca2+ precipitation and uptakes of 14C-labeled substrates were measured by a rapid mixing and filtration procedure. Na-dependent, concentrative uptake was observed for proline, histidine and methylaminoisobutyric acid (MeAIB). Phenylalanine and D-glucose uptakes showed no significant Na dependence. Proline uptake was a saturable function of the proline concentration with Jmax 3.5 nmol/(mg min) and Kt 0.3 mM. Competition experiments in the presence of Na indicated that proline and MeAIB are mutually competitive. Proline uptake was also inhibited 25% by phenylalanine, but MeAIB uptake was relatively unaffected. Neither proline nor MeAIB transport was significantly inhibited by glycine. We conclude that proline uptake across rabbit choroid plexus apical membrane vesicles in via an Na-dependent pathway which is shared by MeAIB and, to a minor extent, by phenylalanine, but from which glycine is excluded. Histidine uptake was inhibited by glycine, GABA, phenylalanine, proline and MeAIB. This suggests that histidine may utilize a different pathway in addition to that shared with proline and MeAIB. These transporters should play an active role in the regulation of amino acids in the CSF.