d-amino acids have recently been found to be present in the extracellular milieu at millimolar levels and are therefore assumed to play a physiological function. However, the pathway (or potential pathways) by which thesed-amino acids are secreted remains unknown. Recently,Escherichia colihas been found to possess one or more energy-dependentd-alanine export systems. To gain insight into these systems, we developed a novel screening system in which cells expressing a putatived-alanine exporter could support the growth ofd-alanine auxotrophs in the presence ofl-alanyl–l-alanine. In the initial screening, fived-alanine exporter candidates, AlaE, YmcD, YciC, YraM, and YidH, were identified. Transport assays of radiolabeledd-alanine in cells expressing these candidates indicated that YciC and AlaE resulted in lower intracellular levels ofd-alanine. Further detailed transport assays of AlaE in intact cells showed that it exportsd-alanine in an expression-dependent manner. In addition, the growth constraints on cells in the presence of 90 mMd-alanine were mitigated by the overexpression of AlaE, implying that AlaE could export freed-alanine in addition tol-alanine under conditions in which intracellulard/l-alanine levels are raised. This study also shows, for the first time, that YciC could function as ad-alanine exporter in intact cells.