Renal glutamate extraction in vivo shows a preference for the uptake of D-glutamate on the antiluminal and L-glutamate on the luminal tubule surface. To characterize this functional asymmetry, we isolated rat kidneys and perfused them with an artificial plasma solution containing either D- or L-glutamate alone or in combination with the system X_AG specific transport inhibitor, D-aspartate. To confirm that removal of glutamate represented transport into tubule cells, we monitored products formed as the result of intracellular metabolism and related these to the uptake process. Perfusion with D-glutamate alone resulted in a removal rate that equaled or exceeded the L-glutamate removal rate, with uptake predominantly across the antiluminal surface; L-glutamate uptake occurred nearly equally across both luminal and antiluminal surfaces. Thus the preferential uptake of D-glutamate at the antiluminal and L-glutamate at the luminal surface confirms the transport asymmetry observed in vivo. Equimolar D-aspartate concentration blocked most of the antiluminal D-glutamate uptake and a significant portion of the luminal L-glutamate uptake, consistent with system X_AG activity at both sites. D-Glutamate uptake was associated with 5-oxo-D-proline production, whereas L-glutamate uptake supported both glutamine and 5-oxo-L-proline formation; D-aspartate reduced production of both 5-oxoproline and glutamine. The presence of system X_AG activity on both the luminal and antiluminal tubule surfaces, exhibiting different reactivity toward L- and D-glutamate suggests that functional asymmetry may reflect two different X_AG transporter subtypes.