We utilized HRP.1 cells derived from midgestation rat placental labyrinth to determine that the primary pathway for glutamate uptake is via system X, a Na⁺-dependent transport system. Kinetic parameters of system X activity were similar to those previously determined in rat and human placental membrane vesicle preparations. Amino acid depletion caused a significant upregulation of system X activity at 6, 24, and 48 h. This increase was reversed by the addition of glutamate and aspartate but not by the addition of -(methylamino)isobutyric acid. Immunoblot analysis of the three transport proteins previously associated with system X activity indicated a trend toward an increase in GLT1, EAAC1, and GLAST1 immunoreactive protein contents by 48 h; cell surface expression of the same was enhanced by 24 h. Inhibition analysis suggested key roles for EAAC1 and GLAST1 in basal anionic amino acid transfer, with an enhanced role for GLT1 under conditions of amino acid depletion. In summary, amino acid availability as well as intracellular metabolism regulate anionic amino acid uptake into this placental cell line.