The Na+-dependent transport of 5-oxoproline into rabbit renal brush-border vesicles was stimulated by a K+ diffusion potential (interior-negative) induced by valinomycin. Na+ salts of two anions of different epithelial permeabilities also affected 5-oxoproline transport. These results show that the Na+-dependent 5-oxoproline transport in renal brush-border vesicles is an electrogenic process which results in a net transfer of positive charge. Maximum transport of 5-oxoproline occurred at an extravesicular pH of 6.0 to 8.0 and over that pH range, 5-oxoproline exists completely as an anion with a negative charge. The simplest stoichiometry consistent with this process is, therefore, the cotransport of one 5-oxoproline anion with two sodium ions. The presence of K+ inside the vesicles stimulated the Na+-dependent transport of 5-oxoproline. This stimulatory effect was specific for K+ and required the presence of Na+. The presence of Na+ gradient was not mandatory for the K+ action. The stimulation by the intravesicular K+ was seen in the presence as well as in the absence of a K+ gradient. Therefore, the increased influx of 5-oxoproline was not coupled to the simultaneous efflux of K+. The presence of K+ in the extravesicular medium alone did not affect the Na+-dependent transport of 5-oxoproline, showing that the site of K+ action was intravesicular. Glutamate did not interact with the Na+-dependent 5-oxoproline transport even in the presence of an outward K+ gradient.