A membrane preparation enriched in the basolateral segment of the plasma membrane was isolated from the rat renal cortex by a procedure that included separation of particulates on a self-generating Percoll gradient. The uptake of L-glutamate by the basolateral membrane vesicles was studied. A Na+ gradient (Na+]o greater than [Na+]i) stimulated the uptake of L-glutamate and provided the driving force for the uphill transport of the acidic amino acid, suggesting a Na+-L-glutamate cotransport system in the basolateral membrane. A K+ gradient ([K+]i greater than [K+]o) increased the uptake additionally. This effect was specific for K+(Rb+). The action of the K+ gradient in enhancing the uptake of L-glutamate had an absolute requirement for Na+. In the presence of Na+, but in the absence of a Na+ gradient. i.e., [Na+]o = [Na+]i, the K+ gradient also energized the concentrative uptake of L-glutamate. This effect of the K+ gradient was not attributable to an alteration in membrane potential. The finding of a concentrative uptake system for L-glutamate energized by both Na+ ([Na+]o greater than [Na+]i and K+ ([K+]o) gradients in the basolateral membrane, combined with previous reports of an ion gradient-dependent uphill transport system for this amino acid in the brush border membrane, suggests a mechanism by which L-glutamate is accumulated intracellularly in the renal proximal tubule to extraordinarily high concentrations.