The organic anions, p-aminohippurate (PAH) and fluorescein, are transported across the basolateral membrane of the renal proximal tubule in exchange for intracellular alpha-ketoglutarate (alpha KG), a mechanism indirectly coupled to sodium via Na+/alpha KG cotransport. To determine whether this mechanism mediates the basolateral transport of other organic anions, transport of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was examined in rat renal cortical slices and basolateral membrane vesicles. In slices, uptake of 2,4-D increased steadily over time, approaching steady-state tissue/medium ratios of approximately 8 after 60 min. Probenecid, PAH and chlorophenol red inhibited steady-state uptake of 2,4-D. Accumulation of 10 microM 2,4-D was stimulated 2-fold by 60 microM glutarate; other dicarboxylic acids failed to stimulate uptake. In the presence of sodium, the addition of 5 mM LiCl or 2 mM ouabain to the bathing medium abolished glutarate stimulation. Removal of sodium from the bathing medium reversibly inhibited uptake as much as 75%. Furthermore, PAH inhibited 2,4-D uptake by slices in a dose-dependent manner, and increasing the external 2,4-D concentration decreased the inhibitory potency of PAH. In basolateral membrane vesicles, unlabeled 2,4-D inhibited sodium glutarate-coupled uptake of 3H-labeled PAH and 2,4-D in a concentration-dependent manner. Moreover, concentrative uptake of 2,4-D into vesicles could be driven by an outwardly directed gradient of glutarate or alpha KG that was generated by lithium-sensitive Na+/dicarboxylate cotransport or imposed experimentally. An outwardly directed gradient of unlabeled 2,4-D or PAH also stimulated uptake of 2,4-D. Based on these data, basolateral accumulation of 2,4-D by the renal proximal tubule is mediated by 2,4-D/alpha KG exchange, a mechanism energetically coupled to Na+/alpha KG cotransport and shared with PAH.